Abstract 2986: Targeting multiple nodes in the RTK- PI3K/AKT/mTOR signaling pathway in a p53-/- mouse model of DIPG induces G2/M phase cell cycle arrest
Authors: Wu Yue Linda , Maachani Uday Bhanu , Schweitzer Melanie , Becher Oren J. , Wang Melinda , Singh Ranjodh , Zhou Zhiping , Souweidane Mark M. 
Published: July 2016

Abstract
Introduction: Diffuse intrinsic pontine glioma (DIPG) is the most common pediatric brainstem tumor, but its prognosis is dismal, with a median survival time of less than one year. Prior studies have implicated amplifications in the receptor tyrosine kinase (RTK)-PI3K/AKT/mTOR signaling pathway in DIPG gliomagenesis, and platelet-derived growth factor receptor (PDGFR) is the most commonly over-expressed RTK. One treatment strategy is thus to inhibit kinases in this pathway with drugs such as dasatinib (PDGFR inhibitor), perifosine (AKT inhibitor), and everolimus (mTOR inhibitor). In this study, we aim to show that combinatorial therapy with dasatinib, perifosine, and everolimus is more effective in impeding tumor cell growth than each drug individually.
Methods: Mouse brainstem glioma cells (mBSG) were derived from a transgenic mouse model of DIPG driven by PDGFB overexpression and p53 loss. Cells were treated for 72 hours with dasatinib, perifosine, and everolimus individually and in combination, and cell viability was assayed with MTS. Western blot with cleaved-caspase 3 antibody was used to assess apoptosis. Cell cycle analysis was performed by propidium iodide flow cytometry.
Results: GI50 concentrations of each individual drug were as follows: 50 nM dasatinib, 50 μM perifosine, and 10 μM everolimus. With combined dasatinib and perifosine treatment, 31.6% of cells survived (p = 0.016 vs. dasatinib alone). The addition of everolimus to dasatinib and perifosine further reduced cell survival to 27.0% (p = 0.011 vs. combinatorial treatment with dasatinib and perifosine). Cell cycle analysis revealed that dasatinib treatment alone prominently arrested cells in G0/G1 phase while both 2-drug combinatorial treatment with dasatinib and perifosine and 3-drug combinatorial treatment with dasatinib, perifosine, and everolimus caused substantial cell cycle arrest in the G0/G1 and G2/M phases. However, Western blot analysis found that no drug treatment group effectively induced apoptosis.
Conclusion: Three-drug combinatorial therapy with dasatinib, perifosine, and everolimus was more effective in reducing DIPG cell viability than dasatinib alone, primarily through the induction of cell cycle arrest at G0/G1 and G2/M. This effect and the lack of apoptosis is consistent with previous observations that inhibition of the PI3K/AKT/mTOR signaling pathway is cytostatic rather than cytotoxic. Moreover, mutations in p53 are seen in up to 50% of patients with DIPG, which allow tumor cells to evade apoptosis despite treatment with chemotherapeutic agents. Taken together, targeting the PI3K/AKT/mTOR pathway alone appears to be insufficient. Ongoing studies aim to understand resistance mechanisms to inhibitors of this pathway and to improve therapeutic efficacy in DIPG by identifying potential synergistic drug combinations targeting alternate survival pathways.
Citation Format: Yue Linda Wu, Uday Bhanu Maachani, Melanie Schweitzer, Oren J. Becher, Melinda Wang, Ranjodh Singh, Zhiping Zhou, Mark M. Souweidane. Targeting multiple nodes in the RTK- PI3K/AKT/mTOR signaling pathway in a p53-/- mouse model of DIPG induces G2/M phase cell cycle arrest. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2986.