Abstract 2431: mTORC1 paradoxically drives MiT/TFE activity and lysosomal biogenesis in tuberous sclerosis complex

Abstract Background: Tuberous Sclerosis Complex (TSC) is characterized by TSC1/2 loss, dysregulated mTORC1 signaling & renal tumors (angiomyolipomas (AML) & renal cell carcinoma (RCC)). The MiT/TFE transcription factors (MITF/TFE3/TFEB) drive autophagy/lysosomal biogenesis & are negatively regulated by mTORC1. However, this model raises a paradox in cancer, where elevated lysosomal activity must persist with mTORC1 activity. We recently showed that epidermal Tsc1 loss paradoxically increases MiT/TFE-activity (https://www.jci.org/articles/view/128287). Intriguingly, TFE3/TFEB gene rearrangements/amplifications and TSC1/2 loss are mutually exclusive drivers in PEComas & RCC. This raises the possibility that TSC1/2 loss & TFE3/TFEB gene rearrangements have overlapping cellular consequences. Here, we address the hypothesis that MiT/TFE-driven lysosomal biogenesis drives tumorigenesis in TSC. Design: We used HEK293T cells +/- CRISPR deletion (KO) of TSC1, 2 or both, to examine: a) Lysosomal gene enrichment by RNA-seq/GSEA, b) Expression of MiT/TFE & lysosomal markers (immunoblotting, qRT-PCR & IF), c) MiT/TFE localization (IF & nuclear fraction immunoblots), d) MiT/TFE activity (4X-CLEAR luciferase assays, qRT-PCR, cathepsin processing, autophagic flux, LC3 puncta). We analysed spontaneous renal tumors in Tsc2 +/- mice for MIT/TFE protein/gene expression (IHC, IF & qRT-PCR analyses of laser capture micro-dissected (LCM) renal tumors). We examined MIT/TFE proteins/lysosomal markers in FFPE samples of renal PEComas & eosinophilic solid & cystic (ESC) RCC & normal kidney. Results: RNA-seq/GSEA showed enrichment of lysosomal gene sets in TSC1/2 KO cells compared to controls. TSC2 KO cells had increased expression of lysosomal transcripts & proteins in cellular lysates & lysosomal fractions. TSC1, 2 & 1/2 KO cells showed increased nuclear TFEB/TFE3 (IF/nuclear-fraction immunoblots), compared to controls. MiT/TFE activity in 4X-CLEAR luciferase reporter assays was increased in TSC2 KO cells compared to controls. Treatment of TSC2 KO cells with chloroquine increased lipidated LC3-II, indicating increased autophagic flux. TSC2 KO cells also showed increased LC3-labelled puncta by IF. We analyzed renal tumors in Tsc2 +/- mice, where elevated mTORC1 signaling was confirmed by p-S6 IHC. Expression of lysosomal proteins (LAMP1, Lamtor 1, Rag C, Cathepsin B) & nuclear localization of TFE3 & TFEB was increased in these lesions by IF/IHC, compared to normal kidney. We performed LCM on renal tumors from Tsc2 +/- mice & found levels of MiT/TFE transcriptional targets to be significantly enriched in tumors compared to normal kidney. We analyzed TFE3 expression in 10 cases of ESC- RCC with sporadic bi-allelic TSC1/2 mutations & 2 cases of TSC-associated RCC; 8/10 cases showed elevated nuclear TFE3. Conclusions: Elevated MiT/TFE levels & activity may represent oncogenic drivers in human & murine renal tumors in TSC. Citation Format: Kaushal V. Asrani, Daniela Salles, Juhyung Woo, Adrianna Mendes, Sanjana Murali, Thiago Vidotto, Pedram Argani, Edward Gabrielson, Tamara Lotan. mTORC1 paradoxically drives MiT/TFE activity and lysosomal biogenesis in tuberous sclerosis complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2431.