Targeting mTORC1/2 by a mTOR Kinase Inhibitor (PP242) induces Apoptosis In AML Cells Under Conditions Mimicking Bone Marrow Microenvironment
Authors: Zeng Zhihong , Shi Yuexi , Tsao Twee , Qiu Yihua , Kornblau Steven M. , Baggerly Keith , Liu Wenbin , Liu Yi , Rommel Christian , Fruman David A. , Andreeff Michael , Konopleva Marina 
Published: October 2019

Abstract
Abstract 778
The prognosis of patients with acute myeloid leukemia (AML) remains poor. Our studies have demonstrated that chemoresistance of AML is not solely due to increased survival signaling in AML cells, but is also enhanced by microenvironment/leukemia interactions. Bone marrow-derived mesenchymal cells (MSC) comprise an essential component of the leukemia bone marrow microenvironment. MSC have the capacity to support normal and malignant hematopoiesis and protect leukemic cells from chemotherapy. We have previously reported that co-culture of AML cells with MSC results in activation of multiple pro-survival signaling pathways in leukemic cells, from which phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling is the key upstream regulator of survival and chemoresistance (Tabe et al., 2007 Cancer Res. 2007). In this study, we investigated the role of mTOR signaling in primary AML cells co-cultured with stroma and in the in vivo leukemia mouse model utilizing a novel TOR kinase inhibitor PP242 (Intellikine, La Jolla, CA). Unlike rapamycin and its analogs, which suppress TORC1 only partially and do not acutely inhibit TORC2, PP242 has been reported to achieve greater inhibition of both TOR complexes, resulting in broader suppression of the PI3K/AKT/TOR signaling in Ph+ B-ALL and T-cell lymphoma (Feldman, et al., PLoS Biol 2009; Janes, et al., Nat Med. 2010). We first employed reverse phase protein array (RPPA) technique profiling of 53 proteins to determine the changes in activation of signaling pathways in leukemic cells from 20 primary AML samples co-cultured with murine stromal line MS-5. Co-culture with stroma resulted in activation of multiple signaling pathways in primary AML cells, inducing upregulation of pAKT(Thr308) in 18, mTOR in 17, pERK(Thr202/204) in 14, and pSTAT3(Ser727) in 12 of the 20 pt samples. This resulted in significant decrease of spontaneous apoptosis in primary AML samples (average 33.7 ± 3.8% annexin V(+) cells in primary AML without co-culture vs. 19.6 ± 3.1% in primary AML co-cultured with MS5, p = 0.027, n = 20). In a next set of experiments, blockade of mTOR signaling with PP242, in a dose dependent fashion, effectively induced apoptosis in primary AML samples (n = 9) cultured with or without stroma: at 60nM, 6.4 ± 1.8% and 8.8 ± 2.4% specific apoptosis (annexin V+), respectively; at 190nM, 10.5% ± 52.8% and 14.9% ± 3.9%; at 560nM, 17.6.9 ± 5.7%; and 21.9 ± 4.9% at 1.67uM, 27.2 ± 6.1% and 27.3 ± 5.8%; at 5uM, 38.8 ± 6.5% and 37.1 ± 7.2%. Importantly, at low nanomolar concentrations, PP242 attenuates the activities of both TORC1 and TORC2, resulting in inhibition of phosphorylation of AKT at S473, S6K at S240/244 and 4EBP1 at T37/46 in both, primary AML cells and most importantly in MSC cultured alone or co-cultured with AML. In the in vivo leukemia mouse model utilizing GFP/luc-labeled Baf3-FLT3/ITD cells, PP242 (60mg/kg/QD gavage) exerted significantly greater anti-leukemia activity compared with TORC1 inhibitor rapamycin (0.1mg/kg/QD IP, p = 0.03). PP242 suppressed leukemia progression as determined by bioluminescence imaging (average luminescence intensity 5.65 ± 1.75 in control vs. average 2.75 ± 0.65 in PP242 group) and significantly extended survival (p = 0.005). In summary, our findings indicate a novel therapeutic strategy to target leukemia within the BM microenvironment through efficient blockade of mTOR/AKT signaling with novel selective TORC kinase inhibitor.
This research is funded by Intellikine.

Disclosures:
Liu: Intellikine: Employment. Rommel:Intellikine: Employment. Fruman:Intellikine: Research Funding. Konopleva:Intellikine: Research Funding.