Accelerated growth of preadipocyte cultures with TSC1 downregulation might be linked to lipoma development and can be reversed by mTOR or PI3K inhibition

Julika Friedrich; Anna Kirstein; Julia Hentschel; Sandy Richter; Henriette Kiep; Maria Arelin; Platzer Konrad; Torsten Schulz; Andreas Merkenschlager; Wieland Kiess; Jamra Rami Abou; Steffi Mayer; Duc Diana Le; Antje Garten

P2-333

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ESPE Abstracts (2024) 98 P2-333

ESPE2024 Poster Category 2 Late Breaking (107 abstracts)

Accelerated growth of preadipocyte cultures with TSC1 downregulation might be linked to lipoma development and can be reversed by mTOR or PI3K inhibition

Julika Friedrich ¹ , Anna Kirstein ¹ , Julia Hentschel ² , Sandy Richter ¹ , Henriette Kiep ³ , Maria Arelin ³ , Platzer Konrad ² , Torsten Schulz ⁴ , Andreas Merkenschlager ³ , Wieland Kiess ³ , Rami Abou Jamra ² , Steffi Mayer ⁵ , Diana Le Duc ² & Antje Garten ¹

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¹University Hospital for Children & Adolescents, Center for Pediatric Research, Leipzig University, Leipzig, Germany. ²Institute of Human Genetics, Leipzig University, Leipzig, Germany. ³University Hospital for Children and Adolescents, Leipzig, Germany. ⁴Department of Orthopedic, Trauma and Plastic Surgery, Leipzig University Hospital, Leipzig, Germany. ⁵University Hospital for Pediatric Surgery, Leipzig, Germany

Background: Tuberous Sclerosis Complex Subunit 1 (TSC1) encodes for the growth inhibitory protein hamartin, which suppresses mTOR signaling. Patients with TSC1 pathogenic variants are prone to developing benign tumors in the brain, kidneys, heart, skin, lungs, and other organs. We identified a likely pathogenic heterozygous germline TSC1 splicing variant NM_000368.5: c.737 +3A>G, r.664_737del, p. (Pro222Valfs*8) in a boy with developmental delay and a large lipoma within the gluteal muscle. We observed a TSC1 loss of heterozygosity in the tumor, which resulted in loss of hamartin protein.

Methods: To assess the effect of TSC1 downregulation, we performed siRNA knockdowns in human preadipocytes (SGBS cells). We measured proliferation by counting nuclei and staining of the proliferation marker Ki67. Pathway activation was assessed by detecting phosphorylated ribosomal protein S6 via Western blot. We quantified adipogenic differentiation via Nile red lipid staining. TSC1 knockdown cells were treated with inhibitors of mTOR (rapamycin and torin-1) and Phosphoinositide-3- (PI3)-kinase (alpelisib).

Results: SGBS cells with TSC1 knockdown showed an increased proliferation, with a 1.7-fold higher cell count on day 7 (P = 0.003) and 7.4-fold increase in the proliferative fraction (Ki67 positive cells) on day 3 (P = 0.0007). Western blots demonstrated higher ribosomal protein S6 phosphorylation in TSC1 KD cells (P = 0.046). There was no effect on adipogenic differentiation capacity of SGBS cells with TSC1 knockdown. Treatment with mTOR and PI3-kinase inhibitors all decreased proliferation and Ki67 positive cells to different extents. Rapamycin decreased cell count by 47 % (P = 0.0004) in the TSC1 KD cells, Torin-1 decreased cell count by 59 % (P = 0.0004) and alpelisib by 49 % (P = 0.0047). While there was no significant difference for torin-1 and alpelisib, rapamycin had stronger effects in the TSC1 KD cells compared to in the control cells (P = 0.015). The proliferative fraction was reduced by 59 % in rapamycin treated cells (P = 0.0096), by 66 % in the torin-1 treated (P = 0.011) and by 63 % in the alpelisib treated cells (P = 0.0054).

Conclusion: Since downregulation of TSC1 led to increased proliferation of SGBS preadipocytes, a causative relationship of the loss-of-function TSC1 variant to lipoma formation is likely in our patient. A tumor regression could not be observed during a 6-month treatment attempt with the rapalog sirolimus. Other rapalogs or PI3-kinase inhibition might be alternative treatment options for the lipoma.