Precision Assessment of on- and Off-Target Effects of mTOR Kinase Inhibitors in a Mouse Model
Authors: Fan Cuiqing , Cai Xiongwei , Zhang Feng , Hochstetler Cindy , Chen Xiaoyi , Guo Fukun , Tian Weidong , Zheng Yi 
Published: October 2019

Abstract
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 analysis also identified ~1000 upregulated genes (such as Ocln, Itgal, and Dlg5) and ~700 downregulated genes (such as Lipg, Cndp2, and Ndst2) in KI vs. KO HSCs: the upregulated genes were enriched in several pathways such as cell adhesion, tight junction, TNF and Ras pathways, while the metabolic pathways were significantly enriched among the downregulated genes. These differential gene expressions underlie the difference in survival between mTOR KI and KO mice, with the median survival for KI mice at 29 days compared to 14 days for KO mice. These results indicate that while the mTOR kinase activity mediates a majority of mTOR regulator pathways, mTOR also confers kinase-independent roles. We next examined gene expression changes of mTOR WT and mTOR KI in response to 200 nM AZD2014. Further examination of differentially expressed (DE) genes between WT+AZD2014 and WT found around 200 DE genes, in which 150 genes (75% changes) overlapped with those DE genes between KI and WT. These overlapped 150 genes shared between WT HSCs treated with AZD2014 and mTOR KI are enriched in a number of important GO and Pathways, such as signal transduction, metabolic pathway, and PI3K-Akt pathway, and they represent on-target effects by the kinase inhibitor AZD2014. On the other hand, less than 40 DE genes between KI+AZD2014 and KI were detected, including Lilrb4, Nptx1 and Ahnak2, which represent off-target genes induced by AZD2014 treatment. Interestingly, the off-target gene set is not enriched in any GO or Pathway. Taken together, our inducible mTOR D2338A KI mouse presents an excellent model for precisely assessing mTOR kinase inhibitor efficacy and specificity. Our studies provide valuable information for the on- and off-target effects by the AZD2014 class of mTOR kinase inhibitors at a therapeutic dose, and reveal several potential biomarkers that can be useful in predicting the off-target effect of mTOR targeted therapies.

Disclosures
No relevant conflicts of interest to declare.