Aurkin-A, a TPX2-Aurora A small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma

Abstract

Chemotherapy induced polyploidy is a mechanism of inherited drug resistance resulting in an aggressive disease course in cancer patients. Alisertib, an Aurora Kinase A (AK-A) ATP site inhibitor, induces cell cycle disruption resulting in polyaneuploidy in Diffuse Large B Cell Lymphoma (DLBCL). Propidium iodide flow cytometry was utilized to quantify alisertib induced polyploidy in U2932 and VAL cell lines. In U2932 cells, 1µM alisertib generated 8n+ polyploidy in 48% of the total cell population after 5 days of treatment. Combination of Aurkin A an AK-A/TPX2 site inhibitor, plus alisertib disrupted alisertib induced polyploidy in a dose-dependent manner with associated increased apoptosis. We generated a stable FUCCI U2932 cell line expressing Geminin-clover (S/G₂/M) and cdt1-mKO (G₁), to monitor cell cycle progression. Using this system, we identified alisertib induces polyploidy through endomitosis, which was eliminated with Aurkin A treatment. In a VAL mouse xenograft model, we show polyploidy generation in alisertib treated mice versus vehicle control or Aurkin A. Aurkin A plus alisertib significantly reduced polyploidy to vehicle control levels. Our in vitro and in vivo studies show that Aurkin A synergizes with alisertib and significantly decreases the alisertib dose needed to disrupt polyploidy while increasing apoptosis in DLBCL cells.

Keywords: Alisertib; Aneuploidy; Cell cycle; Chromosomal instability; Polyploidy.

Graphical abstract

Fig. 1

Alisertib induced polyploidization in Diffuse Large B Cell Lymphoma. U2932 cells treated with DMSO, 50nM, 100nM, 500nM, or 1000nM alisertib for five days were fixed in 70% methanol solution, stained with PI, and analyzed via flow cytometry (A, B). U2932 cells were treated with DMSO (C) or 1µM Alisertib (D). Cells were treated stained with CENP-B and DAPI and imaged at 40x magnification via confocal microscopy.

Fig. 2

Disruption of Alisertib induced polyploidy by Aurkin A. U2932 cells were treated for three or five days with DMSO, Alisertib, Aurkin A, or a combination Alisertib and Aurkin A. Cells were harvested, stained with a propidium iodide solution, and analyzed via flow cytometry (A, B). To assess cellular apoptosis, a PI and FITC Annexin V apoptosis kit was utilized. The FITIC Annex V conjugate will bind to phosphatidylserine present on the plasma membrane of early apoptotic cells (Annexin V positive/PI negative). Necrotic cells with increased cell membrane compromise, will allow PI to enter the cell (Annexin V negative/PI positive). At later stages of apoptosis, phosphatidylserine will be present, along with a high degree of cell membrane comprise, enabling the binding of both Annexin V and PI (double positive, C). Cells that do not uptake the Annex V or PI are considered viable cells. Apoptosis analysis was performed on U2932 cells treated with DMSO, Alisertib, Aurkin A, or a combination of Alisertib and Aurkin A for five days (D). To determine cell viability, MTS assays were performed on U2932 (E), VAL, MDA-MB-231, Hela, Mia PaCa-2, U-2 OS, and Granta-519 cells, treated with either DMSO, Alisertib, Aurkin A, or a combination Alisertib and Aurkin A for four days. Assays were performed in replicates of four and normalized to the DMSO treated condition. Using the Loewe additivity method, the combination index was calculated to determine if the addition of 100µM Aurkin A was synergistic at each inhibitory concentration (F).

Fig. 3

Analysis cell cycle progression using FUCCI U2932 cells. A depicts normal cell cycle progression of FUCCI cells, along with corresponding images of a DMSO control cell. During G1, red fluorescent cdt1-mKO is expressed which transitions to green as Geminin-clover is expressed during S-G2. U2932 cells were treated synchronized with a double thymidine block over a two-day period. Cells were then released from the block by washing with fresh media, supplemented with 1 % final volume DMSO (B).

Fig. 4

Alisertib treatment causes cell cycle disruption and endomitosis. U2932 cells were treated synchronized with a double thymidine block over a two-day period. Cells were then released from the block by washing with fresh media, supplemented with either 500nM alisertib (A) or 1µM alisertib (B). Live fluorescent imaging was performed via an Incucyte SX5 and images were taken every 30 minutes to track cell cycle progression.

Fig. 5

Aurkin A prevents alisertib induced cell cycle slippage and endomitosis. U2932 cells were treated synchronized with a double thymidine block over a two-day period. Cells were then released from the block by washing with fresh media, supplemented with either 100uM Aurkin A (A) or 1µM alisertib and 100µM Aurkin A (B, C). Live fluorescent imaging was performed via an Incucyte SX5 and images were taken every 30 minutes to track cell cycle progression.

Fig. 6

FUCCI cell cycle quantification with AK-A inhibitors. Using the Inucyte Cell-by-Cell analysis software, red and green, fluorescent cells were analyzed (A). Cell-by-Cell was performed on double thymidine synchronized groups treated with DMSO (B), 1µM alisertib (C), 100µM Aurkin A (D), or a combination 1µM alisertib and 100µM Aurkin A (E). All cell groups were performed in five times replicate in groups of 25-50 cells per well.

Fig. 7

Alisertib Induced Polyploidy is Significantly Disrupted by Aurkin A in A VAL Mouse Xenograft Model. Alisertib induced polyploidy is significantly disrupted by Aurkin A in a VAL mouse xenograft model. SCID mice were injected with 1 × 106 VAL cells in sterile saline subcutaneously in the left flank. Mice were pair matched based on tumor volume three weeks post injection and grouped into an alisertib 20mg/kg, Aurkin A 30mg/kg, Alisertib 20mg/kg + Aurkin A 30mg/kg combination, and vehicle control (DMSO volume match) groups. Mice were given the corresponding treatment once a day for five days a week. Treatment was then withheld for two weeks followed by another five-day-a-week treatment cycle. One mouse in the combination group died 15 days after the start of treatment. Tumor volume was normalized to the volume at the start of treatment and graphed. There was a statistically significant difference in tumor volume between the combination group and the control (p=0.0153), alisertib (p=0.027), and Aurkin A (p=0.0323) groups (A). Tumors dissected and fixed in formalin and paraffin embedded. Slides were H&E stained and QuPath Bioimaging Analysis software performed for nucleus to cell ratio to identify polyploidy cells (B). depicts H&E stain from a vehicle control treated mouse. The ploidy classification performed by QuPath highlights normalploid cells in green and polyploid cells in blue (C). Three different fields of at least 10,000 cells were counted for each condition (D).