Significance: The current application of messenger RNA (mRNA)-based technology has largely been confined to liver diseases because of the lack of a specific and efficient extrahepatic in vivo systemic mRNA delivery system. Here, we have developed a library of N-series lipid nanoparticles (LNPs) that could specifically regulate the protein composition of protein corona on the surface of LNPs, which allows specific delivery of mRNA to the lung. We further demonstrated that our lung-targeting LNP could effectively deliver mouse tuberous sclerosis complex 2 ( Tsc2 ) mRNA into TSC2-null cells and restore its function, resulting in enhanced control of tumor burden in a preclinical model of lymphangioleiomyomatosis, a destructive lung disease caused by loss-of-function mutations in the Tsc2 gene.

Birt–Hogg–Dubé (BHD) syndrome is a tumor-suppressor gene disorder characterized by skin tumors, cystic lung disease and renal cell carcinoma. Very little is known about the molecular pathogenesis of BHD. Clinical similarities between BHD and tuberous sclerosis complex (TSC) suggest that the BHD and TSC proteins may function within a common pathway. The TSC proteins inhibit the activity of the mammalian target of rapamycin complex 1 (TORC1), and in Schizosaccharomyces pombe, Bhd and Tsc1/Tsc2 have opposing roles in the regulation of amino-acid homeostasis. We report here that in mammalian cells, downregulation of BHD reduces the phosphorylation of ribosomal protein S6, an indicator of TORC1 activity. To determine whether folliculin, the product of the BHD gene, regulates mammalian target of rapamycin activity in vivo, we generated a mouse with targeted inactivation of the Bhd gene. The mice developed spontaneous oncocytic cysts and tumors composed of cells that resemble the renal cell carcinomas in BHD patients. The cysts and tumors had low levels of phospho-S6. Taken together, these data indicate that folliculin regulates the activity of TORC1, and suggest a new paradigm in which both inappropriately high and inappropriately low levels of TORC1 activity can be associated with renal tumorigenesis.

Tuberous sclerosis complex (TSC) is a tumor suppressor syndrome characterized by benign tumors in multiple organs, including the brain and kidney. TSC-associated tumors exhibit hyperactivation of mammalian target of rapamycin complex 1 (mTORC1), a direct inhibitor of autophagy. Autophagy can either promote or inhibit tumorigenesis, depending on the cellular context. The role of autophagy in the pathogenesis and treatment of the multisystem manifestations of TSC is unknown. We found that the combination of mTORC1 and autophagy inhibition was more effective than either treatment alone in inhibiting the survival of tuberin (TSC2)-null cells, growth of TSC2-null xenograft tumors, and development of spontaneous renal tumors in Tsc2 ^+/− mice. Down-regulation of Atg5 induced extensive central necrosis in TSC2-null xenograft tumors, and loss of one allele of Beclin1 almost completely blocked macroscopic renal tumor formation in Tsc2 ^+/− mice. Surprisingly, given the finding that lowering autophagy blocks TSC tumorigenesis, genetic down-regulation of p62/sequestosome 1 (SQSTM1), the autophagy substrate that accumulates in TSC tumors as a consequence of low autophagy levels, strongly inhibited the growth of TSC2-null xenograft tumors. These data demonstrate that autophagy is a critical component of TSC tumorigenesis, suggest that mTORC1 inhibitors may have autophagy-dependent prosurvival effects in TSC, and reveal two distinct therapeutic targets for TSC: autophagy and the autophagy target p62/SQSTM1.

Lymphangioleiomyomatosis (LAM) is an often fatal disease primarily affecting young women in which tuberin (TSC2)-null cells metastasize to the lungs. The mechanisms underlying the striking female predominance of LAM are unknown. We report here that 17-β-estradiol (E ₂ ) causes a 3- to 5-fold increase in pulmonary metastases in male and female mice, respectively, and a striking increase in circulating tumor cells in mice bearing tuberin-null xenograft tumors. E ₂ -induced metastasis is associated with activation of p42/44 MAPK and is completely inhibited by treatment with the MEK1/2 inhibitor, CI-1040. In vitro, E ₂ inhibits anoikis of tuberin-null cells. Finally, using a bioluminescence approach, we found that E ₂ enhances the survival and lung colonization of intravenously injected tuberin-null cells by 3-fold, which is blocked by treatment with CI-1040. Taken together these results reveal a new model for LAM pathogenesis in which activation of MEK-dependent pathways by E ₂ leads to pulmonary metastasis via enhanced survival of detached tuberin-null cells.

Identifying the mechanisms underlying the regulation of immune checkpoint molecules and the therapeutic impact of targeting them in cancer is critical. Here we show that high expression of the immune checkpoint B7-H3 (CD276) and high mTORC1 activity correlate with immunosuppressive phenotypes and worse clinical outcomes in 11,060 TCGA human tumors. We find that mTORC1 upregulates B7-H3 expression via direct phosphorylation of the transcription factor YY2 by p70 S6 kinase. Inhibition of B7-H3 suppresses mTORC1-hyperactive tumor growth via an immune-mediated mechanism involving increased T-cell activity and IFN-γ responses coupled with increased tumor cell expression of MHC-II. CITE-seq reveals strikingly increased cytotoxic CD38⁺CD39⁺CD4⁺ T cells in B7-H3-deficient tumors. In pan-human cancers, a high cytotoxic CD38⁺CD39⁺CD4⁺ T-cell gene signature correlates with better clinical prognosis. These results show that mTORC1-hyperactivity, present in many human tumors including tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), drives B7-H3 expression leading to suppression of cytotoxic CD4⁺ T cells.

Renal angiomyolipoma is a kidney tumor in the perivascular epithelioid (PEComa) family that is common in patients with Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM) but occurs rarely sporadically. Though histologically benign, renal angiomyolipoma can cause life-threatening hemorrhage and kidney failure. Both angiomyolipoma and LAM have mutations in TSC2 or TSC1. However, the frequency and contribution of other somatic events in tumor development is unknown. We performed whole exome sequencing in 32 resected tumor samples (n = 30 angiomyolipoma, n = 2 LAM) from 15 subjects, including three with TSC. Two germline and 22 somatic inactivating mutations in TSC2 were identified, and one germline TSC1 mutation. Twenty of 32 (62%) samples showed copy neutral LOH (CN-LOH) in TSC2 or TSC1 with at least 8 different LOH regions, and 30 of 32 (94%) had biallelic loss of either TSC2 or TSC1. Whole exome sequencing identified a median of 4 somatic non-synonymous coding region mutations (other than in TSC2/TSC1), a mutation rate lower than nearly all other cancer types. Three genes with mutations were known cancer associated genes (BAP1, ARHGAP35 and SPEN), but they were mutated in a single sample each, and were missense variants with uncertain functional effects. Analysis of sixteen angiomyolipomas from a TSC subject showed both second hit point mutations and CN-LOH in TSC2, many of which were distinct, indicating that they were of independent clonal origin. However, three tumors had two shared mutations in addition to private somatic mutations, suggesting a branching evolutionary pattern of tumor development following initiating loss of TSC2. Our results indicate that TSC2 and less commonly TSC1 alterations are the primary essential driver event in angiomyolipoma/LAM, whereas other somatic mutations are rare and likely do not contribute to tumor development. We performed comprehensive genome analysis of a kidney tumor called angiomyolipoma. These tumors are known to develop in most individuals who have Tuberous Sclerosis Complex (TSC) and those who have sporadic lymphangioleiomyomatosis (LAM), and are seen rarely in the general population. In these angiomyolipomas, we found consistent involvement of the TSC2 and TSC1 genes that are known to cause TSC, but very few (<5 on average) mutations elsewhere in the protein-coding regions. This is in stark contrast to other adult solid tumours that typically harbor hundreds to thousands of such mutations. Our results indicate that genetic alterations in TSC2/TSC1 are the primary and essential driver genetic events for development and progression of renal angiomyolipoma. Analysis of multiple angiomyolipomas from a single patient showed distinct genetic aberrations in the majority of samples, indicating that most of the tumors had developed independently. Branched clonal evolution was evident from the observation of three tumors that shared 2 mutations in addition to mutations private to each. Our results indicate that therapeutic approaches for treatment of patients with angiomyolipoma should focus on the consequences of TSC2/TSC1 loss, including but not limited to mTOR activation.