Abstract
Background: Despite significant gains achieved in the treatment of Burkitt lymphoma (BL), current multi-agent immunochemotherapeutic regimens lead to high rates of acute toxicity, and relapsed/refractory disease still represents a significant hurdle with survival expected in only about 20-30% of such patients. Novel targeted therapeutic approaches are necessary to reduce treatment related toxicity in the up-front setting and improve survival in the relapsed/refractory setting. Analyses of genomic abnormalities in BL have identified increased activation of the PI3K/Akt/mTOR pathway in BL, induced by tonic B-cell receptor signaling and increased expression of Myc induced microRNAs (miRs), as having a significant role in Burkitt lymphomagensis. Additionally, recent reports have implicated higher expression of PI3K activating, Myc induced miRs in pediatric patients with a higher risk of relapse. While focused targeting of PI3K with the PI3K-delta isoform specific inhibitor idelalisib has led to significant activity in indolent B-cell lymphomas, limited activity has been noted in the setting of more aggressive forms. A broader inhibition of both upstream and downstream components of the pathway may exhibit more significant anti-lymphoma activity. To this end, we investigated the in vitro effects of PI3K/Akt/mTOR pathway inhibition with the dual pan-PI3K/mTOR inhibitor Omipalisib (GSK458) in chemotherapy-sensitive and -resistant BL cell line models.
Methods: The in vitro effect of omipalisib was investigated in the BL cell lines Raji, Raji 4RH (chemotherapy-rituximab resistant), Raji 8RH (rituximab resistant), Ramos, and Daudi. Cell viability following exposure to omipalisib alone and in combination with cytotoxic chemotherapeutic agents was analyzed using Cell-Titer Glo and Alamar Blue assays. Apoptosis was analyzed using western blotting for PARP and by flow cytometry with Annexin V-propidium iodide staining. Downstream targets in the PI3K/Akt/mTOR pathway were analyzed using western blotting. Cell cycle analysis was performed by flow cytometry using propidium iodide staining. Synergistic activity of combination exposures was determined by calculation of a combination index using CalcuSyn software.
Results: In vitro exposure of BL cell lines to omipalisib in concentrations ranging from 0.05μM to 50μM for 24, 48 or 72 hours resulted in a dose and time dependent decrease in viable cells with significant activity noted at even low nM concentrations (48 hour IC50 values: Raji=1.2μM, Raji 4RH=0.02μM, Raji 8RH=1.9μM, Ramos=0.01μM, Daudi=0.01μM). Flow cytometry for Annexin V and propidium iodide, after 72 hours of single agent exposure to omipalisib, showed a marked induction of apoptosis in all cell lines. For example, at an omipalisib concentration of 200nM, the percentage of Annexin V positive cells were Raji=40.7%, Raji 4RH=4.4%, Raji 8RH=41.5%, Ramos=59.4% and Daudi=46.9%. Approximately ten-fold higher omipalisib concentrations were required to induce similar degrees of apoptosis in the chemotherapy resistant Raji 4RH cell line compared to chemotherapy sensitive cell lines. Western blotting for downstream targets of the PI3K/Akt/mTOR pathway, including S6 and GSK3Β, showed a reduction in phosphorylation after 30 minutes of exposure to omipalisib in all cell lines. Determination of cell cycle progression following exposure to omipalisib for 72 hours at concentrations ranging from 0.006μM to 25μM showed dose-dependent cell cycle arrest in G1 phase in all cell lines; however the chemotherapy resistant Raji 4RH cells arrested in G2/M at higher concentrations. When BL cells were exposed to omipalisib in combination with either doxorubicin or dexamethasone, synergistic anti-tumor activity was observed in all cell lines tested.
Conclusion: Inhibition of PI3K and mTOR by the dual inhibitor omipalisib suppresses activation of the PI3K/Akt/mTOR pathway leading to impaired BL cell proliferation with G1 cell cycle arrest and induction of apoptosis in chemotherapy-sensitive and -resistant cell line models of BL. Inhibition of the PI3K/Akt/mTOR pathway with omipalisib also increases the in vitro response to cytotoxic chemotherapeutic agents. Our findings note the pre-clinical activity of PI3K/Akt/mTOR pathway inhibition in BL and highlight the relevance of pursuing PI3K/Akt/mTOR pathway inhibition as a potential therapeutic option in BL.
Disclosures
No relevant conflicts of interest to declare.
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