MLN0128, a Second-Generation mTOR Kinase Inhibitor, Disrupts Survival Signaling and Triggers Apoptosis in AML

Abnormal mTOR activation leads to enhanced survival signaling in acute myeloid leukemia (AML) cells. The active site mTOR kinase inhibitors (TOR-KI) represent a promising new approach to target the PI3K/AKT/mTOR pathway. In a previous study, we used a synthetic TOR-KI PP242 to demonstrate that disrupting AKT/mTOR signaling with this class of compounds can effectively target leukemic cells under conditions mimicking bone marrow microenvironment[1]. In the current study, we focused on the inhibitory mechanisms in rare and heterogeneous AML stem cells and on the cellular antagonistic responses after mTORC1/2 blockage using MLN0128, a synthetic TOR-KI currently in clinical development. We found that MLN0128 inhibited cell proliferation and induced apoptosis in AML cells by attenuating mTOR complex 1 and 2 (mTORC1/2) activities, and showed higher anti-leukemia potency than PP242. Using high-throughput time-of-flight mass cytometry (CyTOF)[2], combined with advanced computational analytic software Spanning-Tree Progression of Density-Normalized Events (SPADE)[3] and visualization t-Distribution Stochastic Neighbor Embedding (viSNE)[4], we identified AML subsets co-expressing multiple stem cell markers with high AKT/mTOR activity. MLN0128 selectively targets mTORC1/2 downstream signaling in these cells with minimal effects on other pathways, and lacks the effect on non- leukemic stem cells (LSC). The inhibitory effect of MLN0128 on LSC is positively correlated to that of a number of co-expressing stem cell molecules, and to the baseline expression of and the degree of SCF activation on AKT/mTOR signaling. Using the reverse phase protein array (RPPA) technique, we studied expression and phosphorylation changes in 151 proteins from 24 primary AML samples in response to MLN0128, and revealed several pro-survival pathways may protect from MLN0128-induced cellular stress. We identified four druggable targets that were significantly triggered by MLN0128 inhibition: PARP (that can be targeted by PARP inhibitor olaparib), HDAC3 (vorinostat), p-SRC (dasatinib), and STAT5 (ruxolitinib). Blocking AKT/mTOR signaling and these pro-survival cascades triggered complex responses in AML cell lines and primary AML samples, resulting in an overall increase in apoptosis of AML cells compared to single target inhibition. Among the four combinations, MLN0128 and vorinostat elicited the best responses. In summary, our findings indicate that blocking AKT/mTOR signaling with the mTORC1/2 inhibitor MLN0128 causes growth inhibition and apoptosis in AML cells. High-throughput CyTOF technology revealed that MLN0128 selectively targets primary AML stem cells with high AKT/mTOR activity. RPPA mapping multiple intracellular signaling pathways in primary AML cells in response to mTORC1/2 inhibition allowed us to identify compensatory activation of actionable pro-survival pathways. Combinatorial multi-targeted approaches demonstrated various responses to MLN0128 in AML samples, suggesting that personalized treatment selections will be needed in individual AML cases. Finally, utilization of techniques such as CyTOF or RPPA may assist in profiling intra-cellular and inter-patient variability of responses to targeted agents which likely stem from multiple genetic and epigenetic events within individual tumor, and serve as tools for selection of individualized targeted therapies in AML and other malignancies.

Reference

1. Zeng, Z.H., et al., Targeting of mTORC1/2 by the mTOR kinase inhibitor PP242 induces apoptosis in AML cells under conditions mimicking the bone marrow microenvironment. Blood, 2012. 120(13): p. 2679-2689.

2. Bendall, S.C., et al., Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum. Science, 2011. 332(6030): p. 687-696.

3. Qiu, P., et al., Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE. Nature Biotechnology, 2011. 29(10): p. 886-U181.

4. Amir el, A.D., et al., viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat Biotechnol, 2013. 31(6): p. 545-52.