In vivo characterization of glutamine metabolism identifies therapeutic targets in clear cell renal cell carcinoma.

Targeting metabolic vulnerabilities has been proposed as a therapeutic strategy in renal cell carcinoma (RCC). Here, we analyzed the metabolism of patient-derived xenografts (tumorgrafts) from diverse subtypes of RCC. Tumorgrafts from VHL-mutant clear cell RCC (ccRCC) retained metabolic features of human ccRCC and engaged in oxidative and reductive glutamine metabolism. Genetic silencing of isocitrate dehydrogenase-1 or isocitrate dehydrogenase-2 impaired reductive labeling of tricarboxylic acid (TCA) cycle intermediates in vivo and suppressed growth of tumors generated from tumorgraft-derived cells. Glutaminase inhibition reduced the contribution of glutamine to the TCA cycle and resulted in modest suppression of tumorgraft growth. Infusions with [amide-15N]glutamine revealed persistent amidotransferase activity during glutaminase inhibition, and blocking these activities with the amidotransferase inhibitor JHU-083 also reduced tumor growth in both immunocompromised and immunocompetent mice. We conclude that ccRCC tumorgrafts catabolize glutamine via multiple pathways, perhaps explaining why it has been challenging to achieve therapeutic responses in patients by inhibiting glutaminase.

HIRA loss transforms FH-deficient cells.

Fumarate hydratase (FH) is a mitochondrial enzyme that catalyzes the reversible hydration of fumarate to malate in the tricarboxylic acid (TCA) cycle. Germline mutations of FH lead to hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a cancer syndrome characterized by a highly aggressive form of renal cancer. Although HLRCC tumors metastasize rapidly, FH-deficient mice develop premalignant cysts in the kidneys, rather than carcinomas. How Fh1-deficient cells overcome these tumor-suppressive events during transformation is unknown. Here, we perform a genome-wide CRISPR-Cas9 screen to identify genes that, when ablated, enhance the proliferation of Fh1-deficient cells. We found that the depletion of the histone cell cycle regulator (HIRA) enhances proliferation and invasion of Fh1-deficient cells in vitro and in vivo. Mechanistically, Hira loss activates MYC and its target genes, increasing nucleotide metabolism specifically in Fh1-deficient cells, independent of its histone chaperone activity. These results are instrumental for understanding mechanisms of tumorigenesis in HLRCC and the development of targeted treatments for patients.

The renal lineage factor PAX8 controls oncogenic signalling in kidney cancer.

Large-scale human genetic data1-3 have shown that cancer mutations display strong tissue-selectivity, but how this selectivity arises remains unclear. Here, using experimental models, functional genomics and analyses of patient samples, we demonstrate that the lineage transcription factor paired box 8 (PAX8) is required for oncogenic signalling by two common genetic alterations that cause clear cell renal cell carcinoma (ccRCC) in humans: the germline variant rs7948643 at 11q13.3 and somatic inactivation of the von Hippel-Lindau tumour suppressor (VHL)4-6. VHL loss, which is observed in about 90% of ccRCCs, can lead to hypoxia-inducible factor 2α (HIF2A) stabilization6,7. We show that HIF2A is preferentially recruited to PAX8-bound transcriptional enhancers, including a pro-tumorigenic cyclin D1 (CCND1) enhancer that is controlled by PAX8 and HIF2A. The ccRCC-protective allele C at rs7948643 inhibits PAX8 binding at this enhancer and downstream activation of CCND1 expression. Co-option of a PAX8-dependent physiological programme that supports the proliferation of normal renal epithelial cells is also required for MYC expression from the ccRCC metastasis-associated amplicons at 8q21.3-q24.3 (ref. 8). These results demonstrate that transcriptional lineage factors are essential for oncogenic signalling and that they mediate tissue-specific cancer risk associated with somatic and inherited genetic variants.

Oncometabolites in renal cancer.

The study of cancer metabolism has evolved vastly beyond the remit of tumour proliferation and survival with the identification of the role of 'oncometabolites' in tumorigenesis. Simply defined, oncometabolites are conventional metabolites that, when aberrantly accumulated, have pro-oncogenic functions. Their discovery has led researchers to revisit the Warburg hypothesis, first postulated in the 1950s, of aberrant metabolism as an aetiological determinant of cancer. As such, the identification of oncometabolites and their utilization in diagnostics and prognostics, as novel therapeutic targets and as biomarkers of disease, are areas of considerable interest in oncology. To date, fumarate, succinate, L-2-hydroxyglutarate (L-2-HG) and D-2-hydroxyglutarate (D-2-HG) have been characterized as bona fide oncometabolites. Extensive metabolic reprogramming occurs during tumour initiation and progression in renal cell carcinoma (RCC) and three oncometabolites - fumarate, succinate and L-2-HG - have been implicated in this disease process. All of these oncometabolites inhibit a superfamily of enzymes known as α-ketoglutarate-dependent dioxygenases, leading to epigenetic dysregulation and induction of pseudohypoxic phenotypes, and also have specific pro-oncogenic capabilities. Oncometabolites could potentially be exploited for the development of novel targeted therapies and as biomarkers of disease.

Toll-like receptors 2 and 6 mediate apoptosis and inflammation in ischemic skeletal myotubes.

Critical limb ischemia (CLI) is associated with skeletal muscle damage. However, the pathophysiology of the muscle damage is poorly understood. Toll-like receptors (TLR) have been attributed to play a role in ischemia-induced tissue damage but their role in skeletal muscle damage in CLI is unknown. TLR2 and TLR6 expression was found to be upregulated in skeletal muscle of patients with CLI. In vitro, ischemia led to upregulation of TLR2 and TLR6 by myotubes, and activation of the downstream TLR signaling pathway. Ischemia-induced activation of the TLR signaling pathway led to secretion of the pro-inflammatory cytokine interleukin-6 and muscle apoptosis, which were abrogated by neutralising TLR2 and TLR6 antibodies. Our study demonstrates that TLR2 and TLR6 are upregulated in ischemic muscle and play a role in ischemia-induced muscle damage. Thus, manipulating the TLR pathway locally may be of potential therapeutic benefit.

Ex-vivo normothermic perfusion in renal transplantation: past, present and future.

PURPOSE OF REVIEW: Marginal donor kidneys make up a substantial proportion of transplanted kidneys. Nonetheless, they are more susceptible to injury during procurement and preservation. Normothermic perfusion is an alternative method of organ preservation that can be used to improve the quality, resuscitate, assess and potentially repair the organ. This review provides an up-to-date summary of the role of normothermic perfusion in renal transplantation and what we can expect in the future. RECENT FINDINGS: Normothermic perfusion has been demonstrated to be a feasible and well tolerated method of organ preservation in a series of extended criteria donor kidneys. Furthermore, normothermic perfusion can be used as a quality assessment tool for kidneys pretransplant. In the future, normothermic perfusion could be used to manipulate the organ using gene and stem cell therapies ameliorating renal function after injury. SUMMARY: In the last 5 years, normothermic perfusion has been translated from an experimental laboratory technique into clinical practice with promising results. Normothermic perfusion has demonstrated itself to be multifaceted in its application in renal transplantation. It may improve early graft function and can be used to assess the quality of the kidney before transplantation. The implications of these applications are crucial in maximizing the use of donor organs available in the perpetual climate of organ shortage.