Abstract B39: Multitarget approach against PI3K, Aurora kinase, and BRD4 leads to improved antitumor activity in Myc-overexpressing lymphoma cells
Authors: Park Steven I. , Lin Carolina P. , Foote Michael , Parton Trevor , Darr David B. , Roth Daniel , Bhatt Aadra P. , Dittmer Dirk P. , Sharpless Norman E. , Damania Blossom 
Published: October 2015

Abstract
Background: Myc has proven extremely difficult to target therapeutically. Therefore, we hypothesized that optimal inhibition of several key targetable pathways involved in Myc signaling could overcome this long-standing problem. We identified phosphoinositide 3-kinase (PI3K), aurora kinase (Aurk), and bromodomain protein (BRD)4 as the primary therapeutic targets to counteract Myc deregulation based on strong evidence that these pathways are essential for tumor maintenance in Myc-driven malignancies.
Methods: Cytotoxicity assays using MTS and trypan blue were used to compare levels of drug sensitivity in lymphoma cell lines with high and low Myc mRNA expression. Apoptosis and cell cycle assays were performed using Annexin V and Propidium Iodide staining. Murine xenograft models were used to assess the efficacy and tolerability of single vs. combined inhibition.
Results: Myc-overexpressing Burkitt lymphoma (Raji) cells were treated with various concentrations of small molecule inhibitors against PI3K (BEZ-235), Aurk (MLN-8237), or BRD4 (I-BET-151) for 48 to 72 hours and cell viability was evaluated. BEZ-235, MLN-8237, and I-BET-151 inhibited cell growth individually with IC-50 of 30 nM, 10 nM, and 650 nM, respectively. Dual treatment with BEZ-235/MLN-8237, BEZ-235/I-BET-151, or MLN-8237/I-BET-151 induced more significant cell growth inhibition as compared to treatment with the single agent alone. The combination index (CI) values were less than 1 at various drug concentrations, indicating that different combinations of BEZ-235, MLN-8237, and I-BET-151 were synergistic in terms of inhibitory effect on tumor cell viability. Superior activity of the dual inhibition was also noted in other Myc-overexpressing lymphoma cells, including Ramos and SUDHL4. Combination treatments also increased apoptosis and induced more pronounced cell cycle arrest compared to the single agent treatment alone. We then analyzed protein expression by Western blot in Myc-overexpressing cells treated with various combinations of BEZ-235, MLN-8237, and I-BET-151. Treatment with BEZ-235 in Myc-overexpressing lymphoma cells resulted in reduced phosphorylated levels of the downstream effector RPS6K, which promotes protein translation and proliferation. MLN-8237 reduced the expression of p-HisH3 and p-Aurk while increasing the expression of p-S6K. Treatment with I-BET-151 resulted in significant reduction of Myc expression. Combined treatments had minimal impact on protein expression patterns compared to individual treatments, and the synergistic effect was independent of depletion of cytoplasmic levels of Myc. Lastly, athymic nude mice bearing Ramos lymphoma xenografts were treated with BEZ-235, MLN-8237, I-BET-151, and various dual-combinations of each agent. The mean tumor volumes in mice treated with negative control, BEZ-235, I-BET-151, and MLN-8237 as single agents were 3480, 2364, 2320, and 671 mm3, respectively, at Day 28. Mice treated with BEZ-235/I-BET-151, MLN-8237/I-BET-151, and BEZ-235/MLN-8237 combinations had mean tumor volumes of 1709, 461, and 166 mm3, respectively, at Day 28. The survival rates for mice treated with negative control, BEZ-235, I-BET-151, and MLN-8237 as single agents were 0%, 10%, 10%, and 70%, respectively, at Day 35. The combination of BEZ-235 and MLN-8237 was associated with significant toxicity with 60% of mice dying from weight loss and failure to thrive despite tumor regression. Mice treated with the MLN-8237 and I-BET-151 combination demonstrated the best survival rate of 100% at Day 35.
Conclusion: Our data demonstrated that Myc-overexpressing tumors can be successfully targeted by inhibiting kinases associated with Myc-signaling. Specifically, MLN-8237, a small molecule inhibitor against AURK, induced apoptosis of Myc-overexpressing tumor cells in vitro and showed the most promising anti-tumor activity in mice bearing Myc-overexpressing lymphoma, especially when combined with I-BET-151, a BRD4 inhibitor.
Citation Format: Steven I. Park, Carolina P. Lin, Michael Foote, Trevor Parton, David B. Darr, Daniel Roth, Aadra P. Bhatt, Dirk P. Dittmer, Norman E. Sharpless, Blossom Damania. Multitarget approach against PI3K, Aurora kinase, and BRD4 leads to improved antitumor activity in Myc-overexpressing lymphoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B39.