Abstract
Background: The proto-oncogene Myc is a key regulator of cell growth and survival, and aberrant Myc expression plays a significant role in various tumors, including non-Hodgkin lymphoma (NHL). Myc-associated lymphoma is clinically aggressive, more resistant to standard therapies, and associated with a significantly higher rate of mortality. Novel treatment paradigms are needed to improve survival of patients with Myc-associated NHL. Expression of Aurora Kinase (Aurk) has been associated with Myc, and Aurk is thought to be essential for the maintenance of Myc-driven lymphoma. Aurk is required for assembly of the mitotic spindle and plays key roles in cell proliferation. Amplification and overexpression of Aurk have been observed in various human tumors, including lymphoma, and are frequently associated with tumor progression as well as resistance to chemotherapy. Inhibition of Aurk may overcome resistance to chemotherapy and improve clinical outcomes in patients with Myc-overexpressing lymphoma.
Methods: Cytotoxicity assays using MTS and trypan blue were used to compare levels of drug sensitivity in lymphoma cell lines resistant or sensitive to a conventional chemotherapeutic drug cyclophosphamide. Apoptosis and cell cycle assays were performed using Annexin V and Propidium Iodide staining. The Multiplexed Inhibitor Beads and quantitative Mass Spectrometry (MIB/MS) assays were used to profile kinome changes in response to Aurk inhibition. Murine xenograft models were used to assess the efficacy and tolerability of single vs. combined therapy.
Results: Two Myc-overexpressing cell lines were identified as resistant (Raji) or sensitive (Ramos) to cyclophosphamide, with IC50 of ~ 400 µM and ~ 250 µM, respectively. Raji cells were characterized by increased expression of multidrug resistant protein 1 (MDR1) and mutated p53. There were no significant differences in baseline Aurk or Myc expressions between Raji and Ramos cells. Both cell lines were sensitive to alisertib, an aurora A kinase inhibitor, with maximum cytotoxicity achieved at ~ 100 nM. Combined treatment with alisertib and cyclophosphamide induced more significant cell growth inhibition as compared to treatment with the single agent alone. The combination index (CI) values were less than 1, indicating that alisertib was synergistic to cyclophosphamide in terms of inhibitory effect on tumor cell viability. Alisertib induced apoptosis and pronounced cell cycle arrest, resulting in polyploidy, in Raji cells. Alisertib had little to no effect on Myc, p53, or the total aurora A kinase protein expression in Raji cells although p-Histone-3-Ser10, a downstream target of Aurk, and p-Src levels were significantly decreased at 24 hours of treatment in vitro. Nocodazole-treated cells had reduced p-Aurk level and increased p-Rb as well as increased Mdm2 when treated with alisertib for 24 hours. Athymic nude mice bearing Ramos or Raji lymphoma xenografts were treated with cyclophosphamide, alisertib, or the combination. As expected, all mice bearing Ramos xenograft had complete tumor regression by day 35 of treatment while all mice bearing Raji xenograft had rapid disease progression with median survival of ~ 35 days when treated with cyclophosphamide alone. In contrast, when treated with the combination of cyclophosphamide and alisertib, all mice bearing Raji xenograft had complete regression of tumor by day 35 and had significant improvement in survival (median survival not reached by day 100) compared to the single agent control (p=0.022). Lastly, kinome analysis of Raji xenograft tumors treated with alisertib showed suppression of various kinases involved in Aurk, Src, and PI3K pathways. Western blot of the Raji tumors treated with a prolonged course (25 days) of alisertib showed significant decrease in p-Src and p53 protein levels.
Conclusion: Our data demonstrates that alisertib induces synthetic lethality and overcomes chemoresistance in Myc-overexpressing tumors even in the presence of MDR1 overexpression and p53 mutation. The synergistic effect was largely independent of depletion of cytoplasmic level of Myc. Alisertib, when combined with a conventional chemotherapy drug, induced apoptosis and cell cycle arrest of Myc-overexpressing tumor cells in vitro and showed promising anti-tumor activity in mice bearing chemoresistant Myc-overexpressing lymphoma.
Disclosures
Park: Janssen: Other: travel; Seattle Genetics: Research Funding; Teva: Research Funding.
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