The Investigational Aurora A Kinase Inhibitor Alisertib Exhibits Broad Activity in Preclinical Models of T-Cell Lymphoma and Is Highly Synergistic with Romidepsin
Authors: Zullo Kelly , Guo Yige , Cooke Laurence , Serrano Xavier Jirau , Mangone Michael , Scotto Luigi , Amengual Jennifer E. , Mahadevan Daruka , O'Connor Owen A. 
Published: October 2019

Aurora A kinase (AAK), a serine-threonine protein kinase, regulates mitotic entry, spindle formation, and cytokinesis. Alisertib is a selective AAK investigational inhibitor with demonstrated clinical activity in acute myeloid leukemia, peripheral T-cell lymphoma (PTCL), DLBCL and other hematologic malignancies. Herein we demonstrate the potent cytotoxicity and apoptotic effects of alisertib in a panel of T-cell-derived lymphoma cell-lines (TCL) (CTCL, HTLV+, T-ALL) and B-cell lymphoma cell-lines (DLBCL-ABC, DLBCL-GCB, MCL) alone and in combination with romidepsin.

Single agent concentration and time effect relationships were generated for 8 TCL, 4 DLBCL (2 ABC, 2 GCB) and 4 MCL cell-lines. The mean IC50 of alisertib in TCL was 350 nM (range 100-1000nM) and in B-cell lymphoma lines (DLBCL, MCL) was 200 nM (range 20-300 nM) at 48 hours, measured by growth inhibition. In all cell lines evaluated, there was a consistent 2-log fold decrease in IC50 values at 72 hours, demonstrating the exquisite impact of time on effect. Combination studies evaluating synergy were performed testing schedule, concentration, and time effect relationships. A combinatorial experiment to identify potential synergistic interactions found no synergy with pralatrexate or proteasome inhibitors, but revealed potent synergy with romidepsin. Interestingly, simultaneous exposure of combined alisertib and romidepsin at their IC10, IC20, and IC30 demonstrated marked synergy only in TCL, but not B-cell lymphomas. The best synergy was observed at 72 hours with synergy coefficients ranging from 0.2 to 0.7. This synergistic interaction was restricted to the TCL cell-lines, with no benefit demonstrated in DLBCL or MCL cell lines. (Table 1)

We further evaluated this synergistic interaction through a time lapse experiment (0-72hr) by live cell imaging and found that the combined alisertib and romidepsin treatment in H9, a CTCL cell line, led to failure of cytokinesis leading to apoptosis. Evidence for apoptosis was confirmed for alisertib in combination with romidepsin in the TCL cell-line, H9 and HH after 72 hours of exposure through increased Puma, Caspase 3 and PARP cleavage, and decreased BCL-xL and BCL2 expression; suggesting that cell death was apoptotic. AnnexinV/propridium iodide via FACS analysis confirmed induction of apoptosis. In addition, cell cycle analysis was performed following 24 hour of treatment of romidepsin and alisertib as a single agent and in combination. Alisertib produced a G2/M arrest while the combination of both single agents induced polyploidy (up to 8N). This finding may also complement the increase in apoptosis in the combination treatment versus the single agents. Furthermore, preliminary results from an in-vivoxenograft demonstrate a synergistic interaction amongst the combined treatment of alisertib and romidepsin when compared to control cohort and both single agent cohorts.

These data support the observation that alisertib produces broad single-agent activity in models of TCL and demonstrates marked synergy with romidepsin. Interestingly this effect is restricted to TCL. Mechanistic studies to better understand this synergistic interaction are ongoing. It is hypothesized that the combined alisertib and romidepsin treatment synergistically inhibit the activation of stat-3, leading to apoptosis and inhibition of cellular proliferation through BCL-xL and c-MYC.

Abstract 4493. Table 1: Synergy Coefficients of Alisertib in Combination with Romidepsin at 72 Hours.[1] Drug Combination DND41(T-ALL) J.CAM 1.6(T-ALL) HH(CTCL) H9(CTCL) C5MJ(HTLV+) Romidepsin [IC10-20] + Alisertib 50 nM 0.96 0.81 1.05 1.1 1.53 Romidepsin [IC10-20] + Alisertib 100nM 0.51 0.56 0.68 0.66 0.58 Romidepsin [IC10-20] + Alisertib 1000 nM 0.40 0.20 0.40 0.46 0.70

[1] 1=additive; <1=synergistic; >1= subadditive

Disclosures

Amengual: Acetylon Pharmaceuticals: Research Funding. O'Connor:Millennium Pharmaceuticals: Consultancy; Celgene : Consultancy.