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.

Tuberous sclerosis complex (TSC) is an incurable multisystem disease characterized by mTORC1-hyperactive tumors. TSC1/2 mutations also occur in other neoplastic disorders, including lymphangioleiomyomatosis (LAM) and bladder cancer. Whether TSC-associated tumors will respond to immunotherapy is unknown. We report here that the programmed death 1 coinhibitory receptor (PD-1) is upregulated on T cells in renal angiomyolipomas (AML) and pulmonary lymphangioleiomyomatosis (LAM). In C57BL/6J mice injected with syngeneic TSC2-deficient cells, anti-PD-1 alone decreased 105K tumor growth by 67% (P < 0.0001); the combination of PD-1 and CTLA-4 blockade was even more effective in suppressing tumor growth. Anti-PD-1 induced complete rejection of TSC2-deficient 105K tumors in 37% of mice (P < 0.05). Double blockade of PD-1 and CTLA-4 induced rejection in 62% of mice (P < 0.01). TSC2 reexpression in TSC2-deficient TMKOC cells enhanced antitumor immunity by increasing T cell infiltration and production of IFN-γ/TNF-α by T cells, suggesting that TSC2 and mTORC1 play specific roles in the induction of antitumor immunity. Finally, 1 month of anti-PD-1 blockade reduced renal tumor burden by 53% (P < 0.01) in genetically engineered Tsc2+/- mice. Taken together, these data demonstrate for the first time to our knowledge that checkpoint blockade may have clinical efficacy for TSC and LAM, and possibly other benign tumor syndromes, potentially yielding complete and durable clinical responses.

Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). Transcription factor EB (TFEB), a master regulator of lysosome biogenesis, is negatively regulated by mTORC1 through a RAG GTPase-dependent phosphorylation. Here we show that lysosomal biogenesis is increased in TSC-associated renal tumors, pulmonary lymphangioleiomyomatosis, kidneys from Tsc2+/− mice, and TSC1/2-deficient cells via a TFEB-dependent mechanism. Interestingly, in TSC1/2-deficient cells, TFEB is hypo-phosphorylated at mTORC1-dependent sites, indicating that mTORC1 is unable to phosphorylate TFEB in the absence of the TSC1/2 complex. Importantly, overexpression of folliculin (FLCN), a GTPase activating protein for RAGC, increases TFEB phosphorylation at the mTORC1 sites in TSC2-deficient cells. Overexpression of constitutively active RAGC is sufficient to relocalize TFEB to the cytoplasm. These findings establish the TSC proteins as critical regulators of lysosomal biogenesis via TFEB and RAGC and identify TFEB as a driver of the proliferation of TSC2-deficient cells.