Magdalena E. Tyburczy

Mosaic and Intronic Mutations in TSC1/TSC2 Explain the Majority of TSC Patients with No Mutation Identified by Conventional Testing

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor gene syndrome due to germline mutations in either TSC1 or TSC2. 10–15% of TSC individuals have no mutation identified (NMI) after thorough conventional molecular diagnostic assessment. 53 TSC subjects who were NMI were studied using next generation sequencing to search for mutations in these genes. Blood/saliva DNA including parental samples were available from all subjects, and skin tumor biopsy DNA was available from six subjects. We identified mutations in 45 of 53 subjects (85%). Mosaicism was observed in the majority (26 of 45, 58%), and intronic mutations were also unusually common, seen in 18 of 45 subjects (40%). Seventeen (38%) mutations were seen at an allele frequency < 5%, five at an allele frequency < 1%, and two were identified in skin tumor biopsies only, and were not seen at appreciable frequency in blood or saliva DNA. These findings illuminate the extent of mosaicism in TSC, indicate the importance of full gene coverage and next generation sequencing for mutation detection, show that analysis of TSC-related tumors can increase the mutation detection rate, indicate that it is not likely that a third TSC gene exists, and enable provision of genetic counseling to the substantial population of TSC individuals who are currently NMI.

Mutations in TSC1, TSC2, and MTOR Are Associated with Response to Rapalogs in Patients with Metastatic Renal Cell Carcinoma

Purpose: We examined the hypothesis that mutations in mTOR pathway genes are associated with response to rapalogs in metastatic renal cell carcinoma (mRCC). Experimental Design: We studied a cohort of mRCC patients who were treated with mTOR inhibitors with distinct clinical outcomes. Tumor DNA from 79 subjects was successfully analyzed for mutations using targeted next-generation sequencing of 560 cancer genes. Responders were defined as those with partial response (PR) by RECIST v1.0 or stable disease with any tumor shrinkage for 6 months or longer. Nonresponders were defined as those with disease progression during the first 3 months of therapy. Fisher exact test assessed the association between mutation status in mTOR pathway genes and treatment response. Results: Mutations in MTOR, TSC1, or TSC2 were more common in responders, 12 (28%) of 43, than nonresponders, 4 (11%) of 36 (P = 0.06). Mutations in TSC1 or TSC2 alone were also more common in responders, 9 (21%), than nonresponders, 2(6%), (P = 0.05). Furthermore, 5 (42%) of 12 subjects with PR had mutations in MTOR, TSC1, or TSC2 compared with 4 (11%) of 36 nonresponders (P = 0.03). Eight additional non-mTOR pathway genes were found to be mutated in at least 4 of 79 tumors (5%); none were associated positively with response. Conclusions: In this cohort of mRCC patients, mutations in MTOR, TSC1, or TSC2 were more common in patients who experienced clinical benefit from rapalogs than in those who progressed. However, a substantial fraction of responders (24 of 43, 56%) had no mTOR pathway mutation identified. Clin Cancer Res; 22(10); 2445–52. ©2016 AACR. See related commentary by Voss and Hsieh, p. 2320

Tuberous sclerosis complex neuropathology requires glutamate-cysteine ligase

IntroductionTuberous sclerosis complex (TSC) is a genetic disease resulting from mutation in TSC1 or TSC2 and subsequent hyperactivation of mammalian Target of Rapamycin (mTOR). Common TSC features include brain lesions, such as cortical tubers and subependymal giant cell astrocytomas (SEGAs). However, the current treatment with mTOR inhibitors has critical limitations. We aimed to identify new targets for TSC pharmacotherapy.ResultsThe results of our shRNA screen point to glutamate-cysteine ligase catalytic subunit (GCLC), a key enzyme in glutathione synthesis, as a contributor to TSC-related phenotype. GCLC inhibition increased cellular stress and reduced mTOR hyperactivity in TSC2-depleted neurons and SEGA-derived cells. Moreover, patients’ brain tubers showed elevated GCLC and stress markers expression. Finally, GCLC inhibition led to growth arrest and death of SEGA-derived cells.ConclusionsWe describe GCLC as a part of redox adaptation in TSC, needed for overgrowth and survival of mutant cells, and provide a potential novel target for SEGA treatment.

Tuberous sclerosis complex inactivation disrupts melanogenesis via mTORC1 activation

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor-suppressor gene syndrome caused by inactivating mutations in either TSC1 or TSC2, and the TSC protein complex is an essential regulator of mTOR complex 1 (mTORC1). Patients with TSC develop hypomelanotic macules (white spots), but the molecular mechanisms underlying their formation are not fully characterized. Using human primary melanocytes and a highly pigmented melanoma cell line, we demonstrate that reduced expression of either TSC1 or TSC2 causes reduced pigmentation through mTORC1 activation, which results in hyperactivation of glycogen synthase kinase 3&bgr; (GSK3&bgr;), followed by phosphorylation of and loss of &bgr;-catenin from the nucleus, thereby reducing expression of microphthalmia-associated transcription factor (MITF), and subsequent reductions in tyrosinase and other genes required for melanogenesis. Genetic suppression or pharmacological inhibition of this signaling cascade at multiple levels restored pigmentation. Importantly, primary melanocytes isolated from hypomelanotic macules from 6 patients with TSC all exhibited reduced TSC2 protein expression, and 1 culture showed biallelic mutation in TSC2, one of which was germline and the second acquired in the melanocytes of the hypomelanotic macule. These findings indicate that the TSC/mTORC1/AKT/GSK3&bgr;/&bgr;-catenin/MITF axis plays a central role in regulating melanogenesis. Interventions that enhance or diminish mTORC1 activity or other nodes in this pathway in melanocytes could potentially modulate pigment production.

Nipple Angiofibromas with Loss of TSC2 Are Associated with Tuberous Sclerosis Complex

Nevertheless, our ability to identify meaningful differences between these two populations suggests that this is a reasonable cutpoint. One hypothesis for the increasing incidence rates of early-onset BCC has been increased awareness and skin surveillance. However, lesion size has not decreased over time, as might be anticipated if earlier detection were the underlying cause of increased incidence (Christenson et al., 2005). Our results likewise suggest that early-onset BCC is associated with aggressive histologic characteristics, as opposed to a less aggressive phenotype that might be expected if surveillance bias were operating. Although additional studies are needed, these results suggest there may be underlying biological differences between earlyand late-onset BCC.

Subependymal Giant Cell Astrocytoma: Gene Expression Profiling

Subependymal giant cell astrocytomas (SEGAs) are rare brain tumors occurring in patients with tuberous sclerosis complex (TSC). TSC is characterized by formation of benign tumors in many organs and neurologic disorders (epilepsy, mental retardation, and autism). The disease is caused by mutations in TSC1 (hamartin) or TSC2 (tuberin) which encode tumor suppressors. Inactivation of TSC1/TSC2 leads to enhanced activity of mTOR kinase that regulates i.a. transcription, translation, and cell growth. Data from experiments on lower eukaryotes showed several hundred newly identified genes modulated by TOR at the transcriptional level. However, very little was known about such genes in the human brain. Therefore, we performed global gene expression profiling in SEGAs with use of Affymetrix microarrays, which revealed a considerable number of differentially expressed genes. Identified genes differentially expressed in SEGAs when compared to normal human brain were mainly involved in tumorigenesis (ANXA1, GPNMB, S100A11, LTF, RND3, and SFRP4; up-regulated in SEGA), and the nervous system development and differentiation. Moreover, down-regulation of genes associated with functioning of the nervous system (e.g. CADPS2, CNDP1, ERMIN, GABRA1, MBP, MOBP, NEUROD1, NPTX1, RELN, SHANK3, and TF) in SEGA may be a cause of neurologic dysfunctions in TSC patients. Furthermore, up-regulation of MBP, ERMIN (markers of oligodendrocytes), NEUROD1, and NPTX1 (markers of immature neurons) in SEGA cells compared with normal human astrocytes suggested their mixed, glio-neuronal nature and inability to undergo terminal differentiation. Since SEGAs in patients with TSC appear to be of a mixed glio-neuronal lineage, the current practice of classifying these tumors as astrocytomas merits revision. Finally, it was shown that expression of ANXA1, GPNMB, S100A11, LTF, RND3, SFRP4, and NPTX1 was regulated by mTOR in SEGA cells. The presented data demonstrate new insights into transcriptional regulation induced by mTOR signaling dysfunctions. We anticipate that these results may influence future preclinical and clinical trials for TSC.

Apparent Sporadic Lymphangioleiomyomatosis in a Man as a Result of Extreme Mosaicism for a TSC2 Mutation

1 Division of Pulmonary & Critical Care, University of Michigan, Ann Arbor, MI 2 Department of Dermatology, Uniformed Services University of the Health Sciences, Bethesda, MD 3 Department of Radiology, University of Michigan, Ann Arbor, MI 4 Department of Pathology, University of Michigan, Ann Arbor, MI 5 Division of Pulmonary & Critical Care, University of Cincinnati, Cincinnati, OH 6 Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 7 Brigham and Women’s Hospital, Harvard Medical School, Boston, MA *co-senior authors