Precision Assessment of on- and Off-Target Effects of mTOR Kinase Inhibitors in a Mouse Model

mTOR plays a critical role in controlling cell growth, survival and metabolism and is an important anti-cancer target. There are over 2,000 clinical trials involving mTOR inhibitors as single or combo-agent. The first gen mTOR inhibitors, i.e. rapamycin and rapalogs, are only effective towards mTORC1 and have shown limited efficacy in multiple clinical settings. The second gen mTOR inhibitors target mTOR kinase domain with significantly stronger antineoplastic potency, and over 30 clinical trials of various mTOR kinase inhibitors including AZD2014 are under way. While the new mTOR inhibitors are promising in effectiveness to suppress both mTORC1 and mTORC2 signaling mediated through the mTOR kinase activity, their safety and toxicity features remain a major concern. To precisely determine the on-/off-target effects of mTOR kinase inhibitors, we have generated the conditional Mx1-Cre;mTORflox/flox and Mx1-Cre;mTORflox/knockin D2338Amice that can inducibly yield the mTOR-/- (KO) and the mTOR kinase-activity deficient D2338A mutant knockin (KI) blood genotypes, respectively, upon poly I:C induction. Our previous studies have shown that mTOR KO in blood cells causes hematopoietic failure, with a remarkable reduction in bone marrow cellularity and a transient expansion but long-term exhaustion of hematopoietic stem cells (HSCs). mTOR KO HSCs displayed a loss of quiescence and increased proliferation but normal survival activity. In the current study, we found that the mTOR kinase-deficient D2338A KI mice show several similar phenotypes as mTOR KO, including a drastic inhibition of the mTORC1/mTORC2 downstream effectors p-S6K, p-4E-BP1 and p-AKT (S473) and transient increase in HSC number and proliferation. mTOR kinase deficiency leads to defective engraftment of HSCs in transplantation and failure of colony-formation by progenitors. RNA-seq analysis of the HSC (Lin-Sca-1+c-Kit+CD135-) population reveals that loss of mTOR and loss of mTOR kinase activity share similar changes in over a thousand genes which are enriched in functional clusters including ribosome biogenesis, cell cycle, MAPK pathway, PI3K-Akt pathway, Jak-Stat pathway, and NFkB pathway. Upon mTOR knockout or knockin, several key compensatory genes involved in cell proliferation and survival, including c-myc, Ccnd1, Fos, Jun, and Dusp1 are upregulated. RT-PCR and Western blot further validated that mTOR KO or KI leads to elevated mRNA and protein expression of these compensatory genes. Our RNA-seq a