In Vitro Assessment of Sarcoma Cell Lines Sensitivity to mTOR Inhibitors and Correlation with Genomic Data Evidence Limited Therapeutic Potential
Francois Le Loarer, Gaelle Perot, Pauline Lagarde, Anne Lise Peille, Jean Michel Coindre, Frederic Chibon. Institut Bergonie, Bordeaux, France
Background: Sarcomas with complex genetic profiles account for 40% of sarcomas and display highly rearranged genomic profiles. Large series have highlighted high frequency of 10q deletions encompassing PTEN tumour suppressor gene locus. Pten-deficient murine models develop carcinomas or sarcomas which growth is sustained by AKT-mTOR signaling. In this setting, mTOR inhibitors potently halt tumour growth. Limited therapeutic effects have been evidenced so far in humans but results have rarely been correlated with genomic alterations. We compared in vitro effects of therapies targeting mTOR signaling network in sarcoma cell lines and integrated achieved results with genomic data.
Design: We selected seven fully-annotated sarcoma cell lines harbouring either PTEN deletion (IB105, IB106, MFH148, LPS78) or not (MH100, MFH152, LPS80). Cell lines were exposed for 96h to either a mTOR inhibitor (rapamycin, RAD001 or PP242), or an anti-IGF-1R antibody (R1507 or R7072) or both. Doxorubicin was used as a positive control. We assessed growth curves by flow cytometry and pro-apoptotic effects by Annexin V-propidium iodide staining. mTOR activity was measured by western blot at basal conditions and under treatment with phospho and native antibodies targeting p70S6K and S6RP, 4E-BP1, PRAS40 and AKT.
Results: Despite potent mTOR inhibition seen on western blot, sarcomas cell lines were poorly sensitive to rapalogs (rapamycin and RAD001) or anti-IGF-1R with antiproliferative effects amounting 37% and 11%, respectively. PP242 elicited strong antiproliferative effects (85% in average) together with apoptosis induction at high doses. Strategies combining mTOR inhibitors to anti-IGF-1R did no elicit additive effects. All PTEN-deleted cell lines displayed consistent expression levels of phospho-p70S6K (serine 371) and phospho-S6RP (serine 235). Neither expression levels of mTOR substrates nor PTEN genomic status were predictive of mTOR inhibitors sensitivity. Concurrent genomic alterations proven to affect mTOR signaling were evidenced in all PTEN-deleted cell lines including either AKT1 or AKT3 deletion.
Conclusions: In our study, only PP242 elicited cytotoxic effects in sarcoma cell lines at high doses. However neither PTEN deletion nor mTOR signaling activation were predictive of sensitivity to mTOR inhibitors. Our work provides evidence of limitations to the mTOR addiction model. Based upon genomic data and scientific evidence, we assume that genomic complexity explain these results and jeopardizes mTOR inhibitors's therapeutic potential in sarcomas with complex genetics.
Category: Bone & Soft Tissue
Monday, March 19, 2012 1:00 PM
Poster Session II # 34, Monday Afternoon