Mutations of the tricarboxylic acid cycle (TCA cycle) enzyme fumarate hydratase (FH) cause Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC)1. FH-deficient renal cancers are highly aggressive and metastasise even when small, leading to an abysmal clinical outcome2. Fumarate, a small molecule metabolite that accumulates in FH-deficient cells, plays a key role in cell transformation, making it a bona fide oncometabolite3. Fumarate was shown to inhibit α-ketoglutarate (aKG)-dependent dioxygenases involved in DNA and histone demethylation4,5. However, the link between fumarate accumulation, epigenetic changes, and tumorigenesis is unclear. Here we show that loss of FH and the subsequent accumulation of fumarate elicits an epithelial-to-mesenchymal-transition (EMT), a phenotypic switch associated with cancer initiation, invasion, and metastasis6. We demonstrate that fumarate inhibits Tet-mediated demethylation of a regulatory region of the antimetastatic miRNA cluster6 miR-200ba429, leading to the expression of EMT-related transcription factors and enhanced migratory properties. These epigenetic and phenotypic changes are recapitulated by the incubation of FH-proficient cells with cell-permeable fumarate. Loss of FH is associated with suppression of miR-200 and EMT signature in renal cancer patients, and is associated with poor clinical outcome. These results imply that loss of FH and fumarate accumulation contribute to the aggressive features of FH-deficient tumours.

Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) are associated with a highly malignant form of renal cancer. We combined analytical chemistry and metabolic computational modelling to investigate the metabolic implications of FH loss in immortalized and primary mouse kidney cells. Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione. Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo. Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.

Inhibition of the chemokine receptor CXCR4 in combination with blockade of the PD-1/PD-L1 T cell checkpoint induces T cell infiltration and anticancer responses in murine and human pancreatic cancer. Here we elucidate the mechanism by which CXCR4 inhibition affects the tumor immune microenvironment. In human immune cell-based chemotaxis assays, we find that CXCL12-stimulated CXCR4 inhibits the directed migration mediated by CXCR1, CXCR3, CXCR5, CXCR6, and CCR2, respectively, chemokine receptors expressed by all of the immune cell types that participate in an integrated immune response. Inhibiting CXCR4 in an experimental cancer medicine study by 1-wk continuous infusion of the small-molecule inhibitor AMD3100 (plerixafor) induces an integrated immune response that is detected by transcriptional analysis of paired biopsies of metastases from patients with microsatellite stable colorectal and pancreatic cancer. This integrated immune response occurs in three other examples of immune-mediated damage to noninfected tissues: Rejecting renal allografts, melanomas clinically responding to anti-PD1 antibody therapy, and microsatellite instable colorectal cancers. Thus, signaling by CXCR4 causes immune suppression in human pancreatic ductal adenocarcinoma and colorectal cancer by impairing the function of the chemokine receptors that mediate the intratumoral accumulation of immune cells.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers, and overall survival rates have barely improved over the past five decades. The antimetabolite gemcitabine remains part of the standard of care but shows very limited antitumor efficacy. Ataxia telangiectasia and Rad3-related protein (ATR), the apical kinase of the intra-S-phase DNA damage response, plays a central role in safeguarding cells from replication stress and can therefore limit the efficacy of antimetabolite drug therapies. We investigated the ability of the ATR inhibitor, AZD6738, to prevent the gemcitabine-induced intra-S-phase checkpoint activation and evaluated the antitumor potential of this combination and In PDAC cell lines, AZD6738 inhibited gemcitabine-induced Chk1 activation, prevented cell-cycle arrest, and restrained RRM2 accumulation, leading to the strong induction of replication stress markers only with the combination. Moreover, synergistic growth inhibition was identified in a panel of 5 mouse and 7 human PDAC cell lines using both Bliss Independence and Loewe models. In clonogenic assays, the combination abrogated survival at concentrations for which single agents had minor effects. , AZD6738 in combination with gemcitabine was well tolerated and induced tumor regression in a subcutaneous allograft model of a Kras; Trp53; Pdx-Cre (KPC) mouse cancer cell line, significantly extending survival. Remarkably, the combination also induced regression of a subgroup of KPC autochthonous tumors, which generally do not respond well to conventional chemotherapy. Altogether, our data suggest that AZD6738 in combination with gemcitabine merits evaluation in a clinical trial in patients with PDAC. .

Background Personalised medicine strategies may improve outcomes in pancreatic ductal adenocarcinoma (PDAC), but validation of predictive biomarkers is required. Having developed a clinical trial to assess the ATR inhibitor, AZD6738, in combination with gemcitabine (ATRi/gem), we investigated ATM loss as a predictive biomarker of response to ATRi/gem in PDAC. Methods Through kinase inhibition, siRNA depletion and CRISPR knockout of ATM, we assessed how ATM targeting affected the sensitivity of PDAC cells to ATRi/gem. Using flow cytometry, immunofluorescence and immunoblotting, we investigated how ATRi/gem synergise in ATM-proficient and ATM-deficient cells, before assessing the impact of ATM loss on ATRi/gem sensitivity in vivo. Results Complete loss of ATM function (through pharmacological inhibition or CRISPR knockout), but not siRNA depletion, sensitised to ATRi/gem. In ATM-deficient cells, ATRi/gem-induced replication catastrophe was augmented, while phospho-Chk2-T68 and phospho-KAP1-S824 persisted via DNA-PK activity. ATRi/gem caused growth delay in ATM-WT xenografts in NSG mice and induced regression in ATM-KO xenografts. Conclusions ATM loss augments replication catastrophe-mediated cell death induced by ATRi/gem and may predict clinical responsiveness to this combination. ATM status should be carefully assessed in tumours from patients with PDAC, since distinction between ATM-low and ATM-null could be critical in maximising the success of clinical trials using ATM expression as a predictive biomarker.

An altered response to DNA damage is commonly associated with genomic instability, a hallmark of cancer. Fumarate hydratase (FH) was recently characterised as a DNA repair factor required in non-homologous end-joining (NHEJ) through the local production of fumarate. Inactivating germline mutations in FH cause hereditary leiomyomatosis and renal cell cancer (HLRCC), a cancer syndrome characterised by accumulation of fumarate. Recent data indicate that, in FH-deficient cells, fumarate suppresses homologous recombination DNA repair upon DNA double-strand breaks, compromising genome integrity. Here, we show that FH loss confers resistance to DNA damage caused by ionising radiation (IR), and promotes early mitotic entry after IR in a fumarate-specific manner, even in the presence of unrepaired damage, by suppressing checkpoint maintenance. We also showed that higher levels of DNA damage foci are detectable in untreated FH-deficient cells. Overall, these data indicate that FH loss and fumarate accumulation lead to a weakened G2 checkpoint that predisposes to endogenous DNA damage and confers resistance to IR.

A ldosterone-producing adenomas (APAs) are responsible for ≈5% of hypertension and are probably its most common curable cause. 1 The heterogeneity of these tumors has been highlighted by the landmark discovery of somatic mutations of KCNJ5, followed by others in ATP2B3, ATP1A1, CACNA1D, and CTNNB1. [2][3][4][5] The genotype and histological features delineate 2 overlapping APA subtypes: the common, large KCNJ5 mutant APAs histology is usually similar to cortisol-producing zona fasciculata (ZF) and smaller APAs with other mutations are mainly composed of zona glomerulosa (ZG)-like compact, lipid-depleted cells.The 2 subtypes present other phenotypic differences: for instance, elevated secretion from an APA of hybrid steroids 18-hydroxycortisol and 18-oxocortisol could be considered a fingerprint of KCNJ5 mutation.6 Further differences about the response to aldosterone secretagogues, such as angiotensin II or metoclopramide, have been previously reported. 7,8 Dopamine is one of the main regulators of aldosterone secretion. 9 The 5 known dopamine receptors belong to the G-protein-coupled receptor superfamily and are classified into 2 major subgroups (D1-like and D2-like) according to their structure and opposite action on cAMP production. Activation of the D1-like subfamily members (D1R and D5R) causes an increase of intracellular cAMP levels. The D2-like subfamily includes D2R, D3R, and D4R receptors; their transduction of signal causes a reduction of cAMP production. 10In human adrenal, most attention has been paid to the D2R subtype. This is expressed in normal ZG and mediates dopamine inhibition of aldosterone response to angiotensin II (but not to adrenocorticotropic hormone or potassium) 11,12 and tonic

Deregulated signal transduction and energy metabolism are hallmarks of cancer and both play a fundamental role in tumorigenesis. While it is increasingly recognised that signalling and metabolism are highly interconnected, the underpinning mechanisms of their co-regulation are still largely unknown. Here we designed and acquired proteomics, phosphoproteomics, and metabolomics experiments in fumarate hydratase (FH) deficient cells and developed a computational modelling approach to identify putative regulatory phosphorylation-sites of metabolic enzymes. We identified previously reported functionally relevant phosphosites and potentially novel regulatory residues in enzymes of the central carbon metabolism. In particular, we showed that pyruvate dehydrogenase (PDHA1) enzymatic activity is inhibited by increased phosphorylation in FH-deficient cells, restricting carbon entry from glucose to the tricarboxylic acid cycle. Moreover, we confirmed PDHA1 phosphorylation in human FH-deficient tumours. Our work provides a novel approach to investigate how post-translational modifications of enzymes regulate metabolism and could have important implications for understanding the metabolic transformation of FH-deficient cancers with potential clinical applications.