Izabela A. Malinowska

Regulable neural progenitor-specific Tsc1 loss yields giant cells with organellar dysfunction in a model of tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is a multiorgan genetic disease in which brain involvement causes epilepsy, intellectual disability, and autism. The hallmark pathological finding in TSC is the cerebral cortical tuber and its unique constituent, giant cells. However, an animal model that replicates giant cells has not yet been described. Here, we report that mosaic induction of Tsc1 loss in neural progenitor cells in Tsc1cc Nestin-rtTA+ TetOp-cre+ embryos by doxycycline leads to multiple neurological symptoms, including severe epilepsy and premature death. Strikingly, Tsc1-null neural progenitor cells develop into highly enlarged giant cells with enlarged vacuoles. We found that the vacuolated giant cells had multiple signs of organelle dysfunction, including markedly increased mitochondria, aberrant lysosomes, and elevated cellular stress. We found similar vacuolated giant cells in human tuber specimens. Postnatal rapamycin treatment completely reversed these phenotypes and rescued the mutants from epilepsy and premature death, despite prenatal onset of Tsc1 loss and mTOR complex 1 activation in the developing brain. This TSC brain model provides insights into the pathogenesis and organelle dysfunction of giant cells, as well as epilepsy control in patients with TSC.

Extrarenal perivascular epithelioid cell tumors (PEComas) respond to mTOR inhibition: Clinical and molecular correlates†

Perivascular epithelioid cell tumors (PEComas) are a group of rare mesenchymal tumors that typically show both melanocytic and smooth muscle cell features. Some types of PEComa are seen at high frequency in tuberous sclerosis complex (TSC). The TSC1 and TSC2 genes are commonly mutated in both TSC‐associated and sporadic PEComas, and mTOR signaling pathway activation is also common in these tumors. Preliminary reports have indicated that the mTOR inhibitors sirolimus and related drugs have activity in some patients with non‐TSC‐associated PEComa. Here, we report on the use of these medications in the treatment of five consecutive patients with extrarenal nonpulmonary PEComas seen at one institution. Three complete responses, one partial response and one case of progression were seen. Molecular studies identified TSC2 aberrations in four of these patients, and TFE3 translocation was excluded in the resistant case. A review of all published cases as well as those reported here indicates that partial or complete response was seen in 6 of 11 PEComas, with 5 of 6 having a complete response. These findings highlight the consistent though incomplete activity of mTOR inhibitors in the treatment of PEComas.

Perivascular epithelioid cell tumors (PEComas) harboring TFE3 gene rearrangements lack the TSC2 alterations characteristic of conventional PEComas: further evidence for a biological distinction.

Perivascular epithelioid cell neoplasms (PEComas) are a group of lesions composed of distinctive perivascular epithelioid cells which typically demonstrate both melanocytic and muscular differentiation. This family includes the common renal angiomyolipoma, pulmonary clear cell sugar tumor, lymphangioleiomyomatosis, and less common neoplasms of the soft tissue, gynecologic and gastrointestinal tracts (7, 8, 9, 14, 16, 19). The cells comprising these lesions may be variably epithelioid or spindled in shape, and have variable cytoplasm ranging from clear to eosinophilic. By immunohistochemistry (IHC), PEComas typically express the melanocytic markers HMB45 and Melan-A and the protease Cathepsin K (18), but also typically label for smooth muscle actin and may express desmin. Some members of the PEComa family (specifically angiomyolipoma and lymphangioleiomyomatosis) are seen with high frequency in the genetic syndrome Tuberous Sclerosis Complex (TSC) (16), and a high frequency of syndromic and sporadic PEComas have demonstrated inactivation of the TSC1 or TSC2 genes (12, 20, 21) with subsequent activation of the mammalian target of rapamycin (mTOR) pathway (15). Specifically, mutation in and loss of heterozygosity (LOH) of TSC2 with loss of expression of the protein tuberin, the protein encoded by TSC2, is consistently found in conventional PEComas. TFE3 is a member of the MiT family of transcription factors, which includes MiTF, TFEB, TFEC, and TFE3 (11). TFE3 gene fusions are known to occur in several types of neoplasia. Alveolar soft part sarcoma (ASPS), a rare epithelioid cell soft tissue sarcoma of uncertain histogenesis, characteristically demonstrates a der (17) t(X;17)(p11;q25) resulting in an ASPL-TFE3 gene fusion (17). In addition, a group of recently-described renal cell carcinomas (RCCs) which often occur in children bear various TFE3 gene fusions; these are designated the Xp11 translocation RCC (1, 2, 5, 6). Moreover, a distinctive subgroup of renal cancers in young patients with overlapping features of melanoma, RCC and PEComas have also proven to harbor TFE3 gene fusions (3). Finally, we have recently identified a subgroup of lesions currently characterized as PEComas which, in contrast to conventional PEComas, harbor TFE3 gene fusions (4). Although the number of cases identified are small, distinctive features of these TFE3-rearranged PEComas include a tendency to young age, absence of the association with tuberous sclerosis, predominant alveolar architecture and epithelioid cytology, minimal immunoreactivity for muscle markers, and strong (3+) TFE3 immunoreactivity. In contrast, conventional PEComas frequently have a spindle cell component, typically label for muscle markers, lack strong TFE3 immunoreactivity, and in young patients are frequently associated with tuberous sclerosis. Since conventional PEComas frequently demonstrate TSC2 LOH, and loss of expression of the tuberin protein which this gene encodes (13), we evaluated TFE3-rearranged PEComas for TSC2 LOH and for tuberin expression by IHC. The study cohort consisted of four PEComas previously shown to harbor TFE3 gene fusions (4), and four conventional PEComas which lacked TFE3 alterations. To assess LOH or allelic loss we performed analysis of three microsatellite markers, STR3, KG8 and STR7 in the region of the TSC2 gene, on paraffin-extracted DNA from tumor and normal tissue as described elsewhere (21). IHC was performed by standard techniques using Target Retrieval Solution pH 6.1 (Dako), incubation with anti-tuberin antibody (1:200 dilution, Cell Signaling, #4308), and development with horseradish peroxidase (HRP)-conjugated secondary antibody and DAB (Dako Envision System). Slides were counterstained with hematoxylin. By IHC, all four of the conventional non-TFE3 PEComas demonstrated loss of tuberin protein labeling by immunohistochemistry, with the surrounding normal tissue serving as an internal control (Figure 1, top row). In contrast, all four of the PEComas previously shown to harbor TFE3 gene fusions demonstrated intact, robust tuberin protein labeling (Figure 1, bottom row). In addition, two of the four conventional PEComas showed LOH or allelic loss for one or more TSC2 microsatellite markers (Figure 1, top right), as we and others have seen previously (12, 15, 20). In contrast, none of the four PEComas previously shown to harbor TFE3 gene fusions demonstrated TSC2 LOH (Figure 1, bottom right). Figure1 Hematoxylin and Eosin staining, IHC for tuberin, and TSC2 microsatellite marker analysis is shown for a conventional PEComa (top row), and a TFE3-rearranged PEComa (bottom row). The insets show regions of normal tissue stained for TSC2 from each tumor ... Thus, these observations, while limited in scope due to the limited number of cases available to us, are consistent with our hypothesis that there is a different pathogenetic mechanism in TFE3-rearranged PEComas which does not involve the TSC2 gene through mutation or allelic loss, or other mechanisms of loss of expression. Thus, they suggest that TFE3-rearranged PEComas represent an entity which morphologically overlaps with conventional PEComas, but is biologically distinctive. This concept has clinical translational importance in that mTORC1 inhibitors, such as rapamycin and everolimus, have been shown to be effective in some cases of PEComas (22). If there is no TSC2 gene involvement in TFE3-rearranged PEComas, then these patients may not respond to mTORC1 inhibitors.

Angiomyolipoma Have Common Mutations in TSC2 but No Other Common Genetic Events

Renal angiomyolipoma are part of the PEComa family of neoplasms, and occur both in association with Tuberous Sclerosis Complex (TSC) and independent of that disorder. Previous studies on the molecular genetic alterations that occur in angiomyolipoma are very limited. We evaluated 9 angiomyolipoma for which frozen tissue was available from a consecutive surgical series. Seven of 8 samples subjected to RT-PCR-cDNA sequencing showed mutations in TSC2; none showed mutations in TSC1 or RHEB. Six of the seven mutations were deletions. We searched for 983 activating and inactivating mutations in 115 genes, and found none in these tumors. Similarly analysis for genomic regions of loss or gain, assessed by Affymetrix SNP6.0 analysis, showed no abnormalities. Loss of heterozygosity in the TSC2 region was commonly seen, except in patients with low frequency TSC2 mutations. We conclude that sporadic renal angiomyolipoma usually have mutations in TSC2, but not TSC1 or RHEB, and have no other common genomic events, among those we searched for. However, chromosomal translocations and gene fusion events were not assessed here. TSC2 inactivation by mutation is a consistent and likely necessary genetic event in the pathogenesis of most angiomyolipoma.

Autophagy-Dependent Metabolic Reprogramming Sensitizes TSC2-Deficient Cells to the Antimetabolite 6-Aminonicotinamide

The mammalian target of rapamycin complex 1 (mTORC1) is hyperactive in many human cancers and in tuberous sclerosis complex (TSC). Autophagy, a key mTORC1-targeted process, is a critical determinant of metabolic homeostasis. Metabolomic profiling was performed to elucidate the cellular consequences of autophagy dysregulation under conditions of hyperactive mTORC1. It was discovered that TSC2-null cells have distinctive autophagy-dependent pentose phosphate pathway (PPP) alterations. This was accompanied by enhanced glucose uptake and utilization, decreased mitochondrial oxygen consumption, and increased mitochondrial reactive oxygen species (ROS) production. Importantly, these findings revealed that the PPP is a key autophagy-dependent compensatory metabolic mechanism. Furthermore, PPP inhibition with 6-aminonicotinamide (6-AN) in combination with autophagy inhibition suppressed proliferation and prompted the activation of NF-κB and CASP1 in TSC2-deficient, but not TSC2-proficient cells. These data demonstrate that TSC2-deficient cells can be therapeutically targeted, without mTORC1 inhibitors, by focusing on their metabolic vulnerabilities. Implications: This study provides proof-of-concept that therapeutic targeting of diseases with hyperactive mTORC1 can be achieved without the application of mTORC1 inhibitors. Mol Cancer Res; 12(1); 48–57. ©2013 AACR.

Graded loss of tuberin in an allelic series of brain models of TSC correlates with survival, and biochemical, histological and behavioral features

Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with prominent brain manifestations due to mutations in either TSC1 or TSC2. Here, we describe novel mouse brain models of TSC generated using conditional hypomorphic and null alleles of Tsc2 combined with the neuron-specific synapsin I cre (SynIcre) allele. This allelic series of homozygous conditional hypomorphic alleles (Tsc2(c-del3/c-del3)SynICre(+)) and heterozygote null/conditional hypomorphic alleles (Tsc2(k/c-del3)SynICre(+)) achieves a graded reduction in expression of Tsc2 in neurons in vivo. The mice demonstrate a progressive neurologic phenotype including hunchback, hind limb clasp, reduced survival and brain and cortical neuron enlargement that correlates with a graded reduction in expression of Tsc2 in the two sets of mice. Both models also showed behavioral abnormalities in anxiety, social interaction and learning assays, which correlated with Tsc2 protein levels as well. The observations demonstrate that there are graded biochemical, cellular and clinical/behavioral effects that are proportional to the extent of reduction in Tsc2 expression in neurons. Further, they suggest that some patients with milder manifestations of TSC may be due to persistent low-level expression of functional protein from their mutant allele. In addition, they point to the potential clinical benefit of strategies to raise TSC2 protein expression from the wild-type allele by even modest amounts.

Therapeutic Trial of Metformin and Bortezomib in a Mouse Model of Tuberous Sclerosis Complex (TSC)

Tuberous sclerosis complex (TSC) is a human genetic disorder in which loss of either TSC1 or TSC2 leads to development of hamartoma lesions, which can progress and be life-threatening or fatal. The TSC1/TSC2 protein complex regulates the state of activation of mTORC1. Tsc2+/− mice develop renal cystadenoma lesions which grow progressively. Both bortezomib and metformin have been proposed as potential therapeutics in TSC. We examined the potential benefit of 1 month treatment with bortezomib, and 4 month treatment with metformin in Tsc2+/− mice. Results were compared to vehicle treatment and treatment with the mTORC1 inhibitor rapamycin for 1 month. We used a quantitative tumor volume measurement on stained paraffin sections to assess the effect of these drugs. The median tumor volume per kidney was decreased by 99% in mice treated with rapamycin (p = 0.0004). In contrast, the median tumor volume per kidney was not significantly reduced for either the bortezomib cohort or the metformin cohort. Biochemical studies confirmed that bortezomib and metformin had their expected pharmacodynamic effects. We conclude that neither bortezomib nor metformin has significant benefit in this native Tsc2+/− mouse model, which suggests limited benefit of these compounds in the treatment of TSC hamartomas and related lesions.

Tsc1-Tp53 loss induces mesothelioma in mice, and evidence for this mechanism in human mesothelioma

Mesothelioma is diagnosed in ∼2500 patients in the United States every year, most often arising in the pleural space, but also occurring as primary peritoneal mesothelioma. The vast majority of patients with mesothelioma die of their disease within 3 years. We developed a new mouse model of mesothelioma by bladder or intraperitoneal injection of adenovirus Cre into mice with conditional alleles of each of Tp53 and Tsc1. Such mice began to develop malignant ascites about 6 months after injection, which was due to peritoneal mesothelioma, on the basis of tumor morphology and immunohistochemical staining. Mesothelioma cell lines were established, which showed loss of both Tsc1 and Tp53, with mammalian target of rapamycin complex (mTORC)1 activation. Treatment of mice with malignant ascites due to mesothelioma with rapamycin led to a marked reduction in ascites, extended survival and a 95–99% reduction in the mesothelioma tumor volume, in comparison with vehicle-treated mice. To see whether TSC1/TSC2 loss was a common genetic event in human mesothelioma, we examined nine human mesothelioma cell lines, and found that four of nine showed persistent activation of mTORC1, although none had loss of TSC1 or TSC2. A tissue microarray analysis of 198 human mesothelioma specimens showed that 33% of cases had reduced TSC2 expression and 60% showed activation of mTOR, indicating that mTOR activation is common in human mesothelioma, suggesting that it is a potential therapeutic target.

Similar Trends in Serum VEGF-D Levels and Kidney Angiomyolipoma Responses with Longer Duration Sirolimus Treatment in Adults with Tuberous Sclerosis

Context We have previously shown that serum VEGF-D is elevated at baseline, correlates with kidney angiomyolipoma size at baseline and 12 months, and decreases with sirolimus treatment in adults with tuberous sclerosis complex (TSC). To further investigate the utility of serum VEGF-D for longer term monitoring of TSC kidney disease, we present VEGF-D level results with 24 month follow-up. Objective To compare 24 month VEGF-D levels in two subgroups of sirolimus treated patients (OFF SIROLIMUS AFTER 12 MONTHS or ON SIROLIMUS AFTER 12 MONTHS). Design and Intervention(s) Serum VEGF-D was measured in samples collected from subjects enrolled in a phase 2 multicenter trial evaluating sirolimus for the treatment of kidney angiomyolipomas associated with TSC or TSC/LAM. All participants were treated with sirolimus from 0–12 months. During months 12–24, sirolimus was discontinued in one subgroup. The other subgroup was treated with additional sirolimus. Setting Adult TSC participants were recruited from six clinical sites in the United States (comprehensive TSC clinics, 5; urology clinic, 1). Patients There were 28 TSC patients who completed all 24 months of the study and serum samples were available at 24 months from 18/28 patients. Main Outcome Measure(s) We compared the percent change in VEGF-D levels (baseline to 24 months) in patients from the two treatment subgroups. Results At 24 months, VEGF-D levels decreased by 67% compared with baseline (to 787±426 pg/ml) in the ON SIROLIMUS AFTER 12 MONTHS group versus a 13% decrease (to 2971±4014 pg/ml) in the OFF SIROLIMUS AFTER 12 MONTHS group (p = 0.013, Mann-Whitney test). A similar trend was observed in kidney angiomyolipoma size but not in pulmonary function tests. Conclusions Serum VEGF-D may be useful for monitoring response to treatment with sirolimus and kidney angiomyolipoma size in patients with TSC, but confirmation is needed. Trial Registration Clinical trials.gov NCT00126672.

Aneurysmal Bone Cyst of the Orbit

We report the case of an aneurysmal bone cyst of the left orbital roof in a 12-year-old boy who presented proptosis of the left eye and painless swelling of the left orbital rim. A 3-cm-large tumor developed in less than 3 months, with first visible signs about 2 months after a minor head injury. Radiologically, the lesion was osteolytic and multicystic. The cyst, filled with hematic fluid, was surgically removed via left frontal craniotomy and a simple curettage with high-speed bur. The patient recovered well and has been in good health throughout 14 months of follow-up.