Metformin modulates autophagy in heterozygous and CRISPR-edited <i>TSC2</i> primary fibroblasts

<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Tuberous Sclerosis Complex (TSC) is a genetic disorder caused by variants in TSC1 or TSC2, leading to mTORC1 hyperactivation and autophagy suppression. Although TSC tumorigenesis typically follows a “two-hit” model, the role of TSC2 haploinsufficiency in autophagy regulation remains unclear. We evaluated autophagy markers in haploinsufficient and gene-edited TSC2 primary cells and investigated the role of metformin in modulating autophagy levels.</jats:p> </jats:sec> <jats:sec> <jats:title>Methods</jats:title> <jats:p>Primary fibroblast cultures were obtained from one healthy individual and three from patients carrying heterozygous germline TSC2 variants: the pathogenic variants c.1008T&gt;G and c.4375C&gt;T.A variant of uncertain significance (VUS) c.724A&gt;T. CRISPR/Cas9-RNP editing was used to model loss of heterozygosity (LOH) in cell pools carrying each variant. Cultures were treated with rapamycin, HBSS, metformin, bafilomycin A1, or vehicle controls, and autophagy was assessed by autolysosomes formation by flow cytometry (acridine orange) and autophagosomes immunofluorescence (LC3 and p-S6K).</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>In wild-type cells, only HBSS increased autophagy-positive (acridine orange-positive) cells versus control (15.6% vs. 7.5%; p=0.003). In heterozygous pathogenic cells, rapamycin and metformin increased autophagic cells: c.1008T&gt;G (16.2%, p=0.006; 17.6%, p=0.002) and c.4375C&gt;T (12.5%, p=0.003; 13.3%, p=0.001), versus DMSO controls (9.2% and 7.1%, respectively). VUS c.724A&gt;T cells, with rapamycin increasing autophagic cells (9.74% vs. 6.5%; p=0.0152). In CRISPR-edited cells, all treatments increased the number of autophagic cells compared to the heterozygous cells: c.1008T&gt;G (rapamycin 27.1% vs. 16.7%, p&lt;0.001; metformin 27.2% vs. 17.6%, p&lt;0.001) and c.4375C&gt;T (rapamycin 21.3% vs. 13.1%, p=0.0021; metformin 21.5% vs. 13.6%, p=0.0029). Editing also restored metformin responsiveness in VUS cells (12.5% vs. 8.4%; p=0.0055). Immunochemistry confirmed increased total LC3II and decreased p-S6K across treated cells compared to the control (DMSO).</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>These findings demonstrate that TSC2 haploinsufficiency functionally impairs autophagy prior to second-hit loss. Metformin effectively restores autophagy with phenotypical changes of mTORC1 blockade, highlighting an accessible translational strategy to restore and induce autophagy in TSC cells.</jats:p> </jats:sec>