TFEB drives mTORC1 hyperactivation and kidney disease in Tuberous Sclerosis Complex.

Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, leading to hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and lesions  in multiple organs including lung (lymphangioleiomyomatosis) and kidney (angiomyolipoma and renal cell carcinoma). Previously, we found that TFEB is constitutively active in TSC. Here, we generated two mouse models of TSC in which kidney pathology is the primary phenotype. Knockout of TFEB rescues kidney pathology and overall survival, indicating that TFEB is the primary driver of renal disease in TSC. Importantly, increased mTORC1 activity in the TSC2 knockout kidneys is normalized by TFEB knockout. In TSC2-deficient cells, Rheb knockdown or Rapamycin treatment paradoxically increases TFEB phosphorylation at the mTORC1-sites and relocalizes TFEB from nucleus to cytoplasm. In mice, Rapamycin treatment normalizes lysosomal gene expression, similar to TFEB knockout, suggesting that Rapamycin's benefit in TSC is TFEB-dependent. These results change the view of the mechanisms of mTORC1 hyperactivation in TSC and may lead to therapeutic avenues.

TFEB and TFE3 drive kidney cystogenesis and tumorigenesis.

Birt-Hogg-Dubé (BHD) syndrome is an inherited familial cancer syndrome characterized by the development of cutaneous lesions, pulmonary cysts, renal tumors and cysts and caused by loss-of-function pathogenic variants in the gene encoding the tumor-suppressor protein folliculin (FLCN). FLCN acts as a negative regulator of TFEB and TFE3 transcription factors, master controllers of lysosomal biogenesis and autophagy, by enabling their phosphorylation by the mechanistic Target Of Rapamycin Complex 1 (mTORC1). We have previously shown that deletion of Tfeb rescued the renal cystic phenotype of kidney-specific Flcn KO mice. Using Flcn/Tfeb/Tfe3 double and triple KO mice, we now show that both Tfeb and Tfe3 contribute, in a differential and cooperative manner, to kidney cystogenesis. Remarkably, the analysis of BHD patient-derived tumor samples revealed increased activation of TFEB/TFE3-mediated transcriptional program and silencing either of the two genes rescued tumorigenesis in human BHD renal tumor cell line-derived xenografts (CDXs). Our findings demonstrate in disease-relevant models that both TFEB and TFE3 are key drivers of renal tumorigenesis and suggest novel therapeutic strategies based on the inhibition of these transcription factors.

Seventh BHD international symposium: recent scientific and clinical advancement.

The 7th Birt-Hogg-Dubé (BHD) International Symposium convened virtually in October 2021. The meeting attracted more than 200 participants internationally and highlighted recent findings in a variety of areas, including genetic insight and molecular understanding of BHD syndrome, structure and function of the tumor suppressor Folliculin (FLCN), therapeutic and clinical advances as well as patients' experiences living with this malady.

A substrate-specific mTORC1 pathway underlies Birt-Hogg-Dubé syndrome.

The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates1-3. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy4,5, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB-unlike other substrates of mTORC1, such as S6K and 4E-BP1- is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt-Hogg-Dubé syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma6,7. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt-Hogg-Dubé syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer.

MiT/TFE Family of Transcription Factors, Lysosomes, and Cancer.

Cancer cells have an increased demand for energy sources to support accelerated rates of growth. When nutrients become limiting, cancer cells may switch to nonconventional energy sources that are mobilized through nutrient scavenging pathways involving autophagy and the lysosome. Thus, several cancers are highly reliant on constitutive activation of these pathways to degrade and recycle cellular materials. Here, we focus on the MiT/TFE family of transcription factors, which control transcriptional programs for autophagy and lysosome biogenesis and have emerged as regulators of energy metabolism in cancer. These new findings complement earlier reports that chromosomal translocations and amplifications involving the MiT/TFE genes contribute to the etiology and pathophysiology of renal cell carcinoma, melanoma, and sarcoma, suggesting pleiotropic roles for these factors in a wider array of cancers. Understanding the interplay between the oncogenic and stress-adaptive roles of MiT/TFE factors could shed light on fundamental mechanisms of cellular homeostasis and point to new strategies for cancer treatment.

Transcriptional activation of RagD GTPase controls mTORC1 and promotes cancer growth.

The mechanistic target of rapamycin complex 1 (mTORC1) is recruited to the lysosome by Rag guanosine triphosphatases (GTPases) and regulates anabolic pathways in response to nutrients. We found that MiT/TFE transcription factors-master regulators of lysosomal and melanosomal biogenesis and autophagy-control mTORC1 lysosomal recruitment and activity by directly regulating the expression of RagD. In mice, this mechanism mediated adaptation to food availability after starvation and physical exercise and played an important role in cancer growth. Up-regulation of MiT/TFE genes in cells and tissues from patients and murine models of renal cell carcinoma, pancreatic ductal adenocarcinoma, and melanoma triggered RagD-mediated mTORC1 induction, resulting in cell hyperproliferation and cancer growth. Thus, this transcriptional regulatory mechanism enables cellular adaptation to nutrient availability and supports the energy-demanding metabolism of cancer cells.

Intracellular route and mechanism of action of ERB-hRNase, a human anti-ErbB2 anticancer immunoagent.

Human anti-ErbB2 immunoRNase with Erbicin fused to HP-RNase (ERB-hRNase) is a fully human immunoRNase made up of human pancreatic RNase fused to a human anti-ErbB2 scFv. It binds selectively with high affinity to ErbB2-positive cells, and specifically inhibits their proliferation, in vitro and in vivo. An investigation of its mechanism of action and its intracellular destination has revealed that ERB-hRNase depends on its RNase activity for cytotoxic action; it reaches the cytosol directly from the endosomal compartment; it is inhibited by the cytosolic RNase inhibitor (cRI), but the levels that ERB-hRNase reaches in the cytosol neutralize cRI, thus inducing cell death by apoptosis.