Neurofibromatosis type 1 (NF1) is characterized by cafe-au-lait spots, skinfold freckling, and cutaneous neurofibromas. No obvious relationships between small mutations (<20 bp) of the NF1 gene and a specific phenotype have previously been demonstrated, which suggests that interaction with either unlinked modifying genes and/or the normal NF1 allele may be involved in the development of the particular clinical features associated with NF1. We identified 21 unrelated probands with NF1 (14 familial and 7 sporadic cases) who were all found to have the same c.2970-2972 delAAT (p.990delM) mutation but no cutaneous neurofibromas or clinically obvious plexiform neurofibromas. Molecular analysis identified the same 3-bp inframe deletion (c.2970-2972 delAAT) in exon 17 of the NF1 gene in all affected subjects. The Delta AAT mutation is predicted to result in the loss of one of two adjacent methionines (codon 991 or 992) ( Delta Met991), in conjunction with silent ACA-->ACG change of codon 990. These two methionine residues are located in a highly conserved region of neurofibromin and are expected, therefore, to have a functional role in the protein. Our data represent results from the first study to correlate a specific small mutation of the NF1 gene to the expression of a particular clinical phenotype. The biological mechanism that relates this specific mutation to the suppression of cutaneous neurofibroma development is unknown.

Purpose: Renal angiomyolipomas are a frequent manifestation of tuberous sclerosis and sporadic lymphangioleiomyomatosis (LAM). These disorders are associated with mutations of TSC1 or TSC2 that lead to overactivation of mTOR complex 1 (mTORC1), suggesting an opportunity for targeted therapy by using mTORC1 inhibitors. This study investigated the efficacy and safety of the mTORC1 inhibitor sirolimus for treatment of renal angiomyolipomas in patients with these disorders.Experimental Design: In this multicenter phase 2 nonrandomized open label trial, 16 patients with tuberous sclerosis or sporadic LAM and renal angiomyolipoma(s) were treated with oral sirolimus for up to 2 years. Steady-state blood levels were 3 to 10 ng/mL. The primary outcome was change in size of renal angiomyolipomas measured by MRI and assessed by Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Secondary outcomes included safety, neurocognitive function, and pulmonary function.Results: The response rate, by RECIST criteria, was 50%. Summated angiomyolipoma diameters were reduced in all 16 patients and by 30% or more in eight (all from the per protocol group of 10). Forty-one of 48 angiomyolipomas were smaller at the last measurement than at baseline. Most shrinkage occurred during the first year of treatment. There was little change in pulmonary function. Recall memory improved in seven of eight patients with tuberous sclerosis. Adverse events were consistent with the known toxicities of sirolimus.Conclusions: This study showed sustained regression of renal angiomyolipomas in patients with tuberous sclerosis or sporadic LAM receiving 2 years of sirolimus treatment. Possible effects on pulmonary function and neurocognition require further investigation.

The Warburg effect is a tumorigenic metabolic adaptation process characterized by augmented aerobic glycolysis, which enhances cellular bioenergetics. In normal cells, energy homeostasis is controlled by AMPK; however, its role in cancer is not understood, as both AMPK-dependent tumor-promoting and -inhibiting functions were reported. Upon stress, energy levels are maintained by increased mitochondrial biogenesis and glycolysis, controlled by transcriptional coactivator PGC-1α and HIF, respectively. In normoxia, AMPK induces PGC-1α, but how HIF is activated is unclear. Germline mutations in the gene encoding the tumor suppressor folliculin (FLCN) lead to Birt-Hogg-Dubé (BHD) syndrome, which is associated with an increased cancer risk. FLCN was identified as an AMPK binding partner, and we evaluated its role with respect to AMPKdependent energy functions. We revealed that loss of FLCN constitutively activates AMPK, resulting in PGC-1α-mediated mitochondrial biogenesis and increased ROS production. ROS induced HIF transcriptional activity and drove Warburg metabolic reprogramming, coupling AMPK-dependent mitochondrial biogenesis to HIF-dependent metabolic changes. This reprogramming stimulated cellular bioenergetics and conferred a HIF-dependent tumorigenic advantage in FLCN-negative cancer cells. Moreover, this pathway is conserved in a BHD-derived tumor. These results indicate that FLCN inhibits tumorigenesis by preventing AMPK-dependent HIF activation and the subsequent Warburg metabolic transformation.

Supported by grants from the Tuberous Sclerosis Association, the James Tudor Foundation, and the Welsh Assembly Government for the Wales Gene Park.Drs. Davies and Sampson report receiving grant support and lecture fees from Wyeth. No other potential conflict of interest relevant to this letter was reported.

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Under conditions of reduced tissue oxygenation, hypoxiainducible factor (HIF) controls many processes, including angiogenesis and cellular metabolism, and also influences cell proliferation and survival decisions. HIF is centrally involved in tumour growth in inherited diseases that give rise to renal cell carcinoma (RCC), such as Von HippelLindau syndrome and tuberous sclerosis complex. In this study, we examined whether HIF is involved in tumour formation of RCC in Birt-Hogg-Dube´syndrome. For this, we analysed a Birt-Hogg-Dube´patient-derived renal tumour cell line (UOK257) that is devoid of the BirtHogg-Dube´protein (BHD) and observed high levels of HIF activity. Knockdown of BHD expression also caused a threefold activation of HIF, which was not as a consequence of more HIF1a or HIF2a protein. Transcription of HIF target genes VEGF, BNIP3 and CCND1 was also increased. We found nuclear localization of HIF1a and increased expression of VEGF, BNIP3 and GLUT1 in a chromophobe carcinoma from a Birt-HoggDube´patient. Our data also reveal that UOK257 cells have high lactate dehydrogenase, pyruvate kinase and 3-hydroxyacyl-CoA dehydrogenase activity. We observed increased expression of pyruvate dehydrogenase kinase 1 (a HIF gene target), which in turn leads to increased phosphorylation and inhibition of pyruvate dehydrogenase. Together with increased protein levels of GLUT1, our data reveal that UOK257 cells favour glycolytic rather than lipid metabolism (a cancer phenomenon termed the 'Warburg effect'). UOK257 cells also possessed a higher expression level of the L-lactate influx monocarboxylate transporter 1 and consequently utilized L-lactate as a metabolic fuel. As a result of their higher dependency on glycolysis, we were able to selectively inhibit the growth of these UOK257 cells by treatment with 2-deoxyglucose. This work suggests that targeting glycolytic metabolism may be used therapeutically to treat Birt-Hogg-Dube´-associated renal lesions.

Inappropriate activation of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is common in cancer and has many cellular consequences including elevated endoplasmic reticulum (ER) stress. Cells employ autophagy as a critical compensatory survival mechanism during ER stress. This study utilised drug-induced ER stress through nelfinavir in order to examine ER stress tolerance in cell lines with hyper-active mTORC1 signalling. Our initial findings in wild type cells showed nelfinavir inhibited mTORC1 signalling and upregulated autophagy, as determined by decreased rpS6 and S6K1 phosphorylation, and SQTSM1 protein expression, respectively. Contrastingly, cells with hyper-active mTORC1 displayed basally elevated levels of ER stress which was greatly exaggerated following nelfinavir treatment, seen through increased CHOP mRNA and XBP1 splicing. To further enhance the effects of nelfinavir, we introduced chloroquine as an autophagy inhibitor. Combination of nelfinavir and chloroquine significantly increased ER stress and caused selective cell death in multiple cell line models with hyper-active mTORC1, whilst control cells with normalised mTORC1 signalling tolerated treatment. By comparing chloroquine to other autophagy inhibitors, we uncovered that selective toxicity invoked by chloroquine was independent of autophagy inhibition yet entrapment of chloroquine to acidified lysosomal/endosomal compartments was necessary for cytotoxicity. Our research demonstrates that combination of nelfinavir and chloroquine has therapeutic potential for treatment of mTORC1-driven tumours.

Tuberous sclerosis complex (TSC) is a genetic disorder characterised by highly variable comorbid dysfunction and subsequent morbidity. The mTOR inhibitor everolimus is indicated for the treatment of adult TSC patients with renal angiomyolipomas (AMLs) and for subependymal giant astrocytoma (SEGA) in both adults and children, based on data from the EXIST-1 and EXIST-2 trials. However, due to the historical predominance of everolimus in the oncology setting, some physicians who treat TSC patients may be unfamiliar with everolimus-associated adverse events (AEs) and appropriate management strategies. This article aims to serve as a resource for specialists including nephrologists, paediatricians, neurologists and geneticists who require practical guidance on the management of events such as non-infectious pneumonitis, rash, stomatitis, infections, and renal AEs. Additional consideration is given to drug interactions, hepatic impairment, fertility, and sexual maturation. Since patients with TSC receive clinical benefit from continued therapy, it is important that everolimus-related events are dealt with appropriately through strategies such as dose modification, interruption, the provision of supportive care, regular monitoring, and patient education.