Targeting senescence as a novel pharmacological approach in Lymphangioleiomyomatosis

Orphanet Journal of Rare Diseases 2024, 18(1): P11 Background: Lymphangioleiomyomatosis (LAM) is a multisystem ultra-rare disease that affects women[1]. LAM cells, characterized by the constitutive activation of mTOR (mechanistic Target of Rapamycin) due to the impaired expression of its regulatory proteins hamartin or tuberin, invade the lung, leading to the loss of pulmonary function [2]. Rapamycin is the only drug approved for LAM, but new therapies are needed, since LAM relapses to rapamycin discontinuation[3]. mTOR is a driver of senescence[4], a stress-response process characterized by the inhibition of the cell cycle and by the acquisition of a senescence-associated secretory phenotype (SASP) [5]. Through SASP, senescent cells reinforce their phenotype and promote senescence in neighbouring cells[6]. However, dysregulation of senescence might contribute to disease onset, as SASP boost a proinflammatory microenvironment and the cell cycle arrest limits tissue regeneration[7]. We recently demonstrated that primary tuberin-deficient LAM/TSC cells have senescent features depending on mTOR hyperactivation and, in an in vitro model of LAM microenvironment, they have the capability to induce senescence in tuberin-expressing primary lung fibroblasts (PLFs) through their conditioned medium (CM), with increased secretion of Interleukin(IL)-8[8]. Interestingly, the lysine acetyl transferase p300 was recently identified as epigenetic driver of cellular senescence and it is known to interact with mTOR[9, 10]. Materials and methods: Changing in senescent features, as the increased β-galactosidase positivity and p21 expression, were measured in PLFs grown in the presence of IL-8 and in LAM/TSC cells treated with a monoclonal antibody or with the small molecule SB225002 targeting the IL-8 receptor CXCR2. Then SB225002 was added to PLFs in LAM/TSC CM to counteract senescence induction. In addition, phosphorylation of H2A.X histone and cleaved Caspase 3 were assessed in senescent induced lymphoblastoid cell line (SI-LCLs) derived from controls (HD) and individuals with p300 haploinsufficiency due to germline pathogenetic variants (EP300+/). Results: The addition of IL-8 in PLFs CM increased their senescence proportionally to its concentration. Moreover, blocking CXCR2 impaired the capability of LAM/TSC CM to induce senescence in PLFs. At the same time, targeting CXCR2 hampered senescence in LAM/TSC cells. Finally, the impaired phosphorylation of H2A.X in HD SI-LCLs compared to EP300+/- LCLs confirmed the role of p300 in senescence, suggesting possible avenues for targeting its activity in LAM. Conclusions: Our results indicate that the modulation of senescence targeting mTOR downstream effectors and non-mTOR regulated mechanisms is an intriguing novel pharmacological approach to interfere with the pathological communication in LAM microenvironment with possible future application in LAM therapy.