Targeting mTORC1/2 by a mTOR Kinase Inhibitor (PP242) induces Apoptosis In AML Cells Under Conditions Mimicking Bone Marrow Microenvironment

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.

Dual inhibition of the mTORC1 and mTORC2 signaling pathways is a promising therapeutic target for adult T‐cell leukemia

“…22 These inhibitors have been shown to inhibit mTORC2mediated Akt Ser-473 phosphorylation and Akt signaling, and used as anticancer agents for hematological malignancies including ATL and other cancers. [22][23][24][25][26][27][28][29][30][31][32] We herein show that the PI3K/Akt/mTOR signaling pathway is crucial for the proliferation of ATL cells and that the mTOR inhibitors are cytotoxic to ATL-cell lines and primary ATL samples. Dual mTOR inhibitors such as PP242 and AZD8055, in particular, block the phosphorylation of Akt at Ser-473, strongly induce apoptosis, and inhibit the growth of ATL-cell xenografts in mice.…”

Targeting the mTOR Pathway in Leukemia

“…CXCR4 is a chemokine receptor expressed on hematopoietic stem cells and AML blasts. It plays a key role in stem cell migration and engraftment within the bone marrow microenvironment [Zeng et al, 2012]. When AML cells were cultured in vitro with bone marrow stroma cells, there was upregulation of mTOR, 4EBP1, S6K, Akt, ERK, and STAT3, suggesting that the interaction with the microenvironment plays a major role in activating intracellular pathways [Zeng et al, 2012].…”

“…When AML cells were cultured in vitro with bone marrow stroma cells, there was upregulation of mTOR, 4EBP1, S6K, Akt, ERK, and STAT3, suggesting that the interaction with the microenvironment plays a major role in activating intracellular pathways [Zeng et al, 2012]. The mTOR inhibitor PP242 demonstrated the ability to not only downregulate mTORC1 and mTORC2 activities, but also decreased CXCR4 expression on AML cells and bone marrow stroma cells that were cultured either with or apart from the AML cells [Zeng et al, 2012]. Thus, PP242 was able to disrupt the interaction between the AML cells and the bone marrow microenvironment [Zeng et al, 2012].…”

“…The mTOR inhibitor PP242 demonstrated the ability to not only downregulate mTORC1 and mTORC2 activities, but also decreased CXCR4 expression on AML cells and bone marrow stroma cells that were cultured either with or apart from the AML cells [Zeng et al, 2012]. Thus, PP242 was able to disrupt the interaction between the AML cells and the bone marrow microenvironment [Zeng et al, 2012]. Clinical trials have not yet investigated combined CXCR4 and PI3K/Akt/mTOR inhibition in AML.…”

Targeting the mTORC2 signaling complex in B cell malignancies

“…Besides PI3Kinase inhibitors, there are specific inhibitors that block mTOR function including Rapamycin and its analogs RAD001 (everolimus) and CCI-779 (temsirolimus) which are inhibitors primarily of mTORC1 complex and act via the immunophilin FK506-binding protein (FKBP12) [18]. In addition there are inhibitors of mTORC1/2 and dual inhibitors of both mTORC1 and 2 [19][20][21].…”