Endocrine Abstracts

JOINT1443

Background: Patients with somatic activating variants in the AKT1 gene (Proteus syndrome) show symptoms like segmental overgrowth, increased tumor risk and vascular malformations. AKT is a central signaling molecule in the growth-promoting phosphoinositide-3- kinase (PI3K)/mTOR pathway and different pathway inhibitors are used for cancer therapy or related overgrowth syndromes. While there is no approved therapy for Proteus patients, benefits of treatment with the mTOR inhibitor rapamycin were described. We aim to evaluate the therapeutic potential of rapamycin as well as the PI3K inhibitor alpelisib and the AKT inhibitor capivasertib in a patient-derived cell model.

Methods: Fibroblasts from a 3-year old patient with segmental overgrowth, harboring an activating AKT1 (E17R) variant, were treated with rapamycin, alpelisib, and capivasertib in vitro. We evaluated cell proliferation, viability, and signaling via automated cell counting, WST-1 cell viability assays, and Western blots. Nuclei size was measured using the Spark Cyto multimode imaging plate reader.

Results: While all inhibitors significantly decreased cell count after 72 h of treatment, there were observable differences between mTOR and AKT/PI3K inhibition. Rapamycin reduced growth of the patient cells by 20% already at concentrations of 1 nM, but higher doses could not further reduce proliferation. In contrast, alpelisib reduced proliferation in a dose-dependent manner by 44% at 10 µM and 55% at 50 µM and capivasertib by 23% at 2 µM and 46% at 10 µM. We observed that all inhibitors had direct effects on cell viability already after 1 h of treatment (1 nM rapamycin reduced viability by 8%, 50 µM alpelisib by 56% and 10 µM capivasertib by 12%), indicating an immediate inhibition of cellular metabolism as detected in WST-1 assays. Nuclei size of patient fibroblasts was increased by 8% compared to control fibroblasts, which was reversed by inhibitor treatment. As observed in Western blots, each of the inhibitors effected cellular signaling differentially: While rapamycin was most effective in blocking activation of downstream mTOR signaling components p70S6K and ribosomal protein S6, it increased AKT1 activation. Alpelisib was less effective in blocking mTOR downstream signaling, but highly effective in reducing AKT activation and downstream GSK3β phosphorylation. Capivasertib reduced S6 and GSK3β phosphorylation.

Conclusion: Inhibitors of the PI3K/mTOR pathway effectively reduce growth of patient-derived fibroblasts with an activating AKT1 variant. While rapamycin is effective in nanomolar doses, alpelisib and capivasertib decrease cell growth further. This might indicate a higher efficiency of these drugs in patients with overgrowth related to AKT1 variants.