Abstract A09: Therapeutic targeting of TSC2-deficient cells with Methotrexate: Results of a drug repurposing screen

Autor: Christian V. Baglini, Fuyuhiko Tamanoi, John M. Asara, Jeffrey J. Heard, Alexander J. Valvezan, Menghang Xia, Brendan D. Manning, Srilatha Sakamuru, Hilaire C. Lam, Elizabeth P. Henske, Harilaos Filippakis, Ruili Huang, Christopher M. Austin, Tianmu Wen, Amine Belaid, Nguyen Truong Sinh
Rok vydání: 2017
Předmět:
Zdroj: Clinical Cancer Research. 23:A09-A09
ISSN: 1557-3265
1078-0432
DOI: 10.1158/1557-3265.pmccavuln16-a09
Popis: Tuberous sclerosis complex (TSC) is a multisystem disorder that affects multiple organ systems, including tumors of the brain, heart, kidney, skin and lung. TSC is caused by germline loss-of-function gene mutations in TSC1 or TSC2, which inhibit the mammalian target of rapamycin (mTOR) signaling pathway. Rapalogs are effective cytostatic agents for the treatment of TSC, but continual lifelong therapy is needed. Therapies for TSC that induce a selective cytocidal response in TSC-deficient cells are not currently available, and could have a major clinical impact for TSC patients. We performed a quantitative high-throughput screen (qHTS) of the NCATS Pharmaceutical Collection (NPC) of 2816 compounds, many of which are drugs already approved for other diseases, for their effect on the proliferation of TSC2-deficient ELT3 cells vs. ELT3 cells in which TSC2 was re-expressed. Agents were tested at fifteen concentrations, from 0.6 nM to 46 µM. Six agents selectively inhibited the growth of the TSC2-deficient cells relative to the TSC2-reexpressing cells. One of top inhibitory agents, methotrexate (MTX), a dihydrofolate reductase inhibitor, is FDA-approved, making it a candidate for “repurposing”. In our studies, we confirmed the selective inhibitory effect of MTX on patient-derived TSC2-deficient cells. Flow cytometry analysis demonstrated that MTX (5 µM, 24 h) arrested 60.8% of the TSC2-deficient cells in S-phase, vs. 26.6% of the TSC2-expressing cells. The accumulation of S-phase arrested cells ultimately leads to apoptosis selectively in the TSC2-deficient cells, which was detected by flow cytometry of Propidium Iodide/Annexin V stained cells (52.3% of apoptosis in TSC2-deficient cells vs. 11% in TSC2-reexpressing cells) and immunoblotting for cleaved Caspase3 and cleaved PARP. Co-treatment with the mTOR inhibitor Rapamycin and MTX prevented MTX-induced apoptosis (52.3% apoptotic cells with MTX alone vs. 11.8% with MTX plus Rapamycin). In vivo, mice bearing TSC2-deficient ELT3 cell xenograft tumors were treated with MTX (25mg/kg or 50mg/kg) or rapamycin (1mg/kg) by intraperitoneal injection every other day over 3 weeks. We found that 50mg/kg of MTX significantly decreased the volume of tumors within two weeks of treatment, to a similar extent as Rapamycin treatment did. We conclude that Methotrexate acts as a selective cytotoxic agent in TSC2-deficient cells. This is consistent with recent discoveries that mTOR regulates the de novo purine and pyrimidine synthesis pathways. As an FDA-approved agent, Methotrexate has potential for clinical “repurposing” for the treatment of TSC-deficient tumors. Citation Format: Amine Belaid, Harilaos Filippakis, Hilaire Lam, Alexander Valvezan, Ruili Huang, Menghang Xia, Tianmu Wen, Christian Baglini, Srilatha Sakamuru, John Asara, Nguyen Truong Sinh, Jeffrey J. Heard, Fuyuhiko Tamanoi, Christopher Austin, Brendan Manning, Elizabeth P. Henske. Therapeutic targeting of TSC2-deficient cells with Methotrexate: Results of a drug repurposing screen. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A09.
Databáze: OpenAIRE
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