ABSTRACTPHENOTYPIC CHARACTERIZATION OF ALLELIC VARIANTS OF THE MECHANISTIC TARGET OF RAPAMYCIN (mTOR)ByJoy GarymTOR is a serine/threonine kinase at the hub of multiple signaling pathways, with roles in proliferation, translation, and growth. The PI3K/AKT/mTOR pathway is frequently hyperactivated in cancers and can be targeted pharmacologically. To better understand the role of mTOR in cancer treatment and in response to cell stressors, mouse models of decreased mTOR and of a single nucleotide polymorphism (SNP) in Mtor were evaluated. Specifically, mice with T-cell-specific, constitutively-active AKT (Lck-MyrAkt) that develop spontaneous thymic lymphomas were crossed to mice with genetically reduced mTOR expression (knock down, KD). Genetic mTOR reduction was associated with prolonged survival (24 weeks in KD mice versus 14 weeks in WT mice), though both eventually developed pre-T lymphoblastic leukemia/lymphoma (pre-T LBL). Transcriptional profiling of the murine pre-T LBL revealed that mTOR KD was associated with decreased expression of Cdk6, a critical proliferative control node in T-cell development and oncogenic transformation. The combination of a mTOR inhibitor (rapamycin) and a CDK4/6 inhibitor (PD-0332991, palbociclib) cooperatively decreased viability and signaling downstream of drug targets in murine thymic lymphoma cells and human T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) cell lines. In addition, the role of mTOR in response to cell stressors was examined in the context of inflammatory, genotoxic, and oncogenic stress. In this study, a rare SNP in Mtor (C1977T, leading to the amino acid substitution R628C), found in BALB/c mice, was linked to decreased DNA damage response (DDR) through gene expression profiling in an inflammatory environment. Mice with the SNP (628C KI) had significantly decreased survival post total body irradiation (TBI) compared to WT and heterozygous mice. Mouse embryonic fibroblasts (MEFs) and bone marrow from 628C KI mice were more sensitive to DNA damage and the DNA damage proteins FANCD2 and ATM were decreased in KI MEFs. Downstream targets of mTOR, most notably those phosphorylated by mTORC2, were also decreased in KI MEFs, especially the target PKCα. Proliferation was decreased by irradiation in WT MEFs, and was minimally affected by irradiation in KI MEFs. KI mice exposed to fractionated irradiation over several weeks developed thymic lymphomas more rapidly and had decreased survival compared to WT mice. Finally, to evaluate oncogenic stress, keratinocytes from KI mice were Ras-transformed and grafted onto nude mice, resulting in a higher rate of papilloma development than seen with keratinocytes from WT mice. The mTOR KD mouse model confirmed the importance of mTOR in T-cell leukemia/lymphoma, while the KI model highlighted the role of mTOR in response to cell stressors that put selective pressure on cancer cells. These findings emphasize the continued importance of research into therapies targeting mTOR and mTOR’s role in cancer progression.
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