Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM) are caused by inactivating mutations in TSC1 or TSC2 , leading to mTORC1 hyperactivation. The mTORC1 inhibitors rapamycin and analogs (rapalogs) are approved for treating of TSC and LAM. Due to their cytostatic and not cytocidal action, discontinuation of treatment leads to tumor regrowth and decline in pulmonary function. Therefore, life-long rapalog treatment is proposed for the control of TSC and LAM lesions, which increases the chances for the development of acquired drug resistance. Understanding the signaling perturbations leading to rapalog resistance is critical for the development of better therapeutic strategies. We developed the first Tsc2-null rapamycin-resistant cell line, ELT3-245, which is highly tumorigenic in mice, and refractory to rapamycin treatment. In vitro ELT3-245 cells exhibit enhanced anchorage-independent cell survival, resistance to anoikis, and loss of epithelial markers. A key alteration in ELT3-245 is increased β-catenin signaling. We propose that a subset of cells in TSC and LAM lesions have additional signaling aberrations, thus possess the potential to become resistant to rapalogs. Alternatively, when challenged with rapalogs TSC-null cells are reprogrammed to express mesenchymal-like markers. These signaling changes could be further exploited to induce clinically-relevant long-term remissions.

Despite the identification of mTOR hyperactivation as the main biochemical defect downstream of TSC1/TSC2 inactivation in Lymphangioleiomyomatosis (LAM) and Tuberous Sclerosis Complex (TSC), and the approval of rapamycin and rapalogs for the treatment of the related lesions, these drugs are cytostatic and continued treatment is required for clinical benefit. The latter raises the possibility of acquired drug resistance over long-term use. To explore the mechanisms leading to rapamycin resistance in LAM/TSC, we xenografted SCID mice with ELT3 cells (Tsc2-null derived from an Eker rat uterine leiomyoma), explanted a tumor that was not responsive to rapamycin, and generated cell line ELT3-245. The cell growth of ELT3-245 is not inhibited by rapamycin in vitro. SCID mice inoculated with ELT3-245 form palpable tumors significantly faster, compared to ELT3 cells, respond very poorly to rapamycin at the initial stages of treatment, but soon become non-responsive. In addition, these cells form macroscopically visible metastases to the lungs of ELT3-245 tumor-bearing mice. We performed global gene expression profiling, comparing ELT3-245 to ELT3 cells, and found that the rapamycin-resistant cells have decreased expression of genes associated with epithelial cells and increased expression of genes associated with mesenchymal-like characteristics. Relative gene expression of Myc, Egfr, Akt2, Jun, and Ocln were assessed by RT-qPCR in ELT3 vs ELT3-245 after 24h of treatment with 20 nM rapamycin or DMSO. Important discrepancies include a 2.8-fold increased expression of Egfr under rapamycin treatment in ELT3 cells but not in ELT3-245; however, ELT3-245 had a 15-fold higher expression of Egfr prior to treatment, compared to ELT3. In addition, ELT3-245 cells strikingly increase expression of Myc when challenged with rapamycin, compared to ELT3. Similarly, Akt2 is increased considerably under rapamycin treatment in ELT3-245 and is already higher prior to treatment compared to ELT3. Finally, Ocln expression was at least 100-fold reduced in ELT3-245, compared to ELT3, both under rapamycin treatment and no-treatment conditions. These results suggest that Myc, Egfr, Akt2, and Ocln are among the genes highly relevant to acquired rapamycin resistance in this Tsc2-null rat leiomyoma cell line and point toward the pathways that need to be interfered with in order to overcome it. Citation Format: Natalia Filippidou, Mathildi Valianou, Daniel L. Johnson, John J. Bissler, Aristotelis Astrinidis. Dissecting rapamycin resistance in a Tsc2-null rat leiomyoma cell line developed in a murine xenograft [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3022.