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A mouse model for assessing mTOR kinase-independent role and off-target effect of mTOR kinase inhibitors [III]

ABSTRACT: mTOR is a serine/threonine kinase with a crucial role in regulating fundamental cellular processes and is an attractive anticancer target. Multiple clinical trials of mTOR kinase inhibitors such as AZD2014 that suppress both mTORC1 and mTORC2 are ongoing, but their specificity and toxicity features remain a concern. We have developed an inducible kinase-dead D2338A mTOR mouse knock-in model and show that it is useful to study the kinase-independent function of mTOR and for assessing the off-target effects of mTOR kinase inhibitors. We found that while the mTOR KI mice in hematopoietic system displayed similar phenotypes to that of mTOR KO mice, they have significantly prolonged survival time than the KO mice. Whereas AZD2014 was mostly on-target in suppressing mTOR kinase activity, mimicking that seen in KI stem cells in transcriptome analysis, off-target genes in KI cells induced by AZD2014 including Ahnak2, Emp1, Lilrb4, Tpsb2, and Cma1 were detected. Our studies reveal kinase-independent function of mTOR in hematopoiesis and presents a mouse model for precisely assessing specificity of mTOR kinase inhibitors. Stringent test of drugs with such mouse models in the preclinical setting may provide useful information guiding the safety and efficacy of related clinical trials.

ORGANISM(S): Mus musculus

PROVIDER: GSE235097 | GEO | 2023/08/29

REPOSITORIES: GEO

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Project description:The mTOR (mammalian Target of Rapamycin) pathway is constitutively activated in Diffuse Large B-Cell Lymphoma (DLBCL). mTOR inhibition has been shown to have clinical activity in patients with DLBCL, although overall response rates remain low. We therefore evaluated differences in the transcriptome between DLBCL cell lines with differential sensitivity to the mTOR inhibitor Rapamycin, to (A) identify gene-expression patterns(GEP) capable of identifying sensitivity to Rapamycin, (B) understand the underlying mechanisms of resistance to Rapamycin in DLBCL and (C) identify bioactive molecules likely to synergize with mTOR inhibitors. Using Affymetrix HuGene ST 1.0 microarrays, we were able to identify a gene expression signature capable of accurately predicting sensitivity and resistance to Rapamycin in DLBCL cell lines. Pathway analysis identified the serine/threonine kinase Akt as central to the differentially-expressed gene network. Connectivity mapping of our datasets identified compounds targeting the AKT pathway with a high likelihood of reversing the GEP associated with resistance to Rapamycin. Specifically, we evaluated the HIV protease inhibitor (PI) Nelfinavir, which is known to have anti-cancer and Akt-inhibitory properties, as well as the small molecule Akt inhibitor MK-2206, for their potential to synergize with to Rapamycin in DLBCL. Nelfinavir and MK-2206 caused profound inhibition of cell viability in combination with Rapamycin in DLBCL cell lines. Low nanomolar concentrations of Rapamycin inhibited phosphorylation of Akt and also downstream targets of activated mTOR when used in combination with these Akt inhibitors. These findings have the potential to significantly improve patient selection for mTOR inhibitor therapy, and to improve rates and depths of response. More broadly, they support the use of global RNA expression and connectivity mapping to improve patient selection and identify synergistic drug combinations for cancer therapy. DLBCL cell lines were tested for Rapamycin sensitivity and classified as "sensitive" or "resistant." Genome-wide analysis of all cell lines were performed using the Affymetrix HuGene ST 1.0 Array Platform. Genes with differential expression between sensitive and resistant cell lines were analyzed using Statistical Analysis of Microarrays (SAM) software, and a signature of genes determnined. This signature was found to accurately predict sensitivity or resistance of other DLBCL cell lines, and to identify the protein kinase Akt as central to resistance.

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A mouse model for assessing mTOR kinase-independent role and off-target effect of mTOR kinase inhibitors [III]

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