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Functional Genomics of the Lysosome

Periodic Reporting for period 4 - LYSOSOMICS (Functional Genomics of the Lysosome)

Reporting period: 2021-04-01 to 2022-03-31

Lysosomes are membrane-enclosed organelles with an important role in regulating cellular metabolism. Dysregulation of lysosomal function is, indeed, causative of metabolic imbalance and is associated with an onset of pathological features including growth dysfunction and inflammation. These phenotypic manifestations are implicated in different metabolic human diseases like cancer and monogenic diseases, like the Lysosomal Storage Disorders (LSDs). The final goal of LYSOSOMICS was a deep understanding of the biological mechanisms by which lysosomes can exert their function as metabolic regulators and how their dysfunction may contribute to cellular detrimental effects associated with human metabolic disease. We proposed the achievement of two main objectives: 1) the Identification of conditions and novel lysosome-mediated pathways that influence specific aspects of lysosomal function, 2) The develop of cellular models for lysosomal diseases for the identification of new therapeutic strategies. Reaching these two objectives we aimed to generate ground-breaking discoveries and find new molecular targets for the development of novel therapeutic strategies to treat human metabolic diseases, such as cancer or LSDs.
For decades the lysosome has been known as the main hub for cellular catabolic and recycling processes. More recent studies expanded this view revealing that major cellular signaling processes occur at the lysosomal surface and play a key role in physiological processes such as nutrient sensing and pathological conditions such as cancer. Recently, we and others showed that two molecules play a fundamental role in orchestrating the lysosomal response to environmental cues: the transcription factor TFEB, a master regulator of the transcriptional program controlling lysosomal function, and the kinase complex mTORC1, a master regulator of cellular metabolism. Over the last decade, we have contributed to elucidate the mechanisms by which TFEB activation is modulated by mTORC1. We discovered that TFEB activity are regulated by the mTORC1 kinase through a lysosome-to-nucleus signaling mechanism (Settembre et al. EMBO J. 2012).This regulatory pathway allows the lysosome to respond to environmental cues as nutrients deprivation and a variety of stress conditions. We also have identified a feedback loop mechanism by which TFEB is not only a substrate but also a regulator of mTORC1, by promoting the expression of proteins that are responsible for the mTORC1 activation.
One of the first goal achieved by LYSOSOMICS has been the identification of a novel signaling pathway, which we named “non-canonical mTORC1 signaling”, by which lysosomes can elicit selective responses to diverse metabolic signals regulating TFEB activity.
We revealed that this newly discovered pathway plays a crucial role in Birt–Hogg–Dubé syndrome, a rare inherited cancer syndrome characterized by the formation of kidney cysts and cancer progression. We found that dysfunction of non-canonical mTORC1 signaling in BHD leads to the development of these phenotypes. Notably, by correcting this pathway in BHD mouse models, we were able to rescue the kidney phenotype that characterizes the disease, thus opening the way to the development of new ways of intervention for the treatment of this devastating disease. The above-mentioned results were recently published in Nature (Napolitano et al. Nature 2020). In addition, our results allowed other investigators to demonstrate that dysfunction of the non-canonical mTORC1 pathway identified by our lab also plays a major pathogenic role in the inherited disorder Tuberous sclerosis (Alesi et al. Nat Commun, 2021) and other diseases (Napolitano et al. Trends Cell Biol, 2022). Our main goal is now to pharmacologically target this pathway for therapeutic intervention. Another important achievement reached by LYSOSOMICS has been the generation of genetically modified cell lines as a model to study Lysosomal Storage Disorders (LSDs), another class of metabolic human diseases caused by lysosomal dysfunction, for which current therapeutic options are inefficient or not available. We generated a biobank of genetically engineered cellular models that recapitulate the pathological environment of LSDs, characterized by lysosomal and cellular dysfunction. We have used these cells for an in-depth analysis of the cellular abnormalities observed in LSDs. In addition, these cellular tools have been made available to the scientific community and have been already distributed to several scientists working on LSDs, who used this tool for the characterization of lysosomal gene function and as a platform for the implementation of new therapeutic approaches (Soldati et al. EMBO Mol Med. 2021; De Risi et al Nat Commun. 2021).
LYSOSOMICS allowed the discovery of a “non-canical” signaling pathway by which the mTORC1 complex can orchestrate cellular response to specific metabolic stimuli by regulating TFEB activation.
We revealed that dysfunction of this mechanism is the main driver of the phenotypes observed in Birt–Hogg–Dubé syndrome (Napolitano et al. Nature 2020), opening a new prospective for the therapeutic treatment of such disease. In addition, the identification of non-canonical mTORC1 signaling has paved the way to novel important discoveries on how cell metabolism is modulated in both normal and pathological conditions. Finally, LYSOSOMICS allowed the generation of a biobank of LSD cellular models, which have already used by us and other scientists as a unique tool for the study of LSD cellular abnormalities and for the implementation of new therapeutic approaches. We expected to accomplish the final analysis of different LSDs models, in particular the Batten disease (CLN3 KO) and Vici Syndrome (EPG5 KO) models, to clarify the specific function of these two genes and their role in the pathological events that underline these two diseases.
LSD phenotypes