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, 20 (10), 965-974

Dual BRD4 and AURKA Inhibition Is Synergistic Against MYCN-Amplified and Nonamplified Neuroblastoma

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Dual BRD4 and AURKA Inhibition Is Synergistic Against MYCN-Amplified and Nonamplified Neuroblastoma

Joshua Felgenhauer et al. Neoplasia.

Abstract

A majority of cases of high-risk neuroblastoma, an embryonal childhood cancer, are driven by MYC or MYCN-driven oncogenic signaling. While considered to be directly "undruggable" therapeutically, MYC and MYCN can be repressed transcriptionally by inhibition of Bromodomain-containing protein 4 (BRD4) or destabilized posttranslationally by inhibition of Aurora Kinase A (AURKA). Preclinical and early-phase clinical studies of BRD4 and AURKA inhibitors, however, show limited efficacy against neuroblastoma when used alone. We report our studies on the concomitant use of the BRD4 inhibitor I-BET151 and AURKA inhibitor alisertib. We show that, in vitro, the drugs act synergistically to inhibit viability in four models of high-risk neuroblastoma. We demonstrate that this synergy is driven, in part, by the ability of I-BET151 to mitigate reflexive upregulation of AURKA, MYC, and MYCN in response to AURKA inhibition. We then demonstrate that I-BET151 and alisertib are effective in prolonging survival in four xenograft neuroblastoma models in vivo, and this efficacy is augmented by the addition of the antitubule chemotherapeutic vincristine. These data suggest that epigenetic and posttranslational inhibition of MYC/MYCN-driven pathways may have significant clinical efficacy against neuroblastoma.

Figures

Figure 1
Figure 1
I-BET151 and alisertib are synergistic in their effects on neuroblastoma cell viability in vitro. Combination Index (CI) plots are shown for NB1643 (A), NB-SD (B), SK-N-SH (C), and SK-N-AS (D) neuroblastoma cell lines. Cells were treated with a combination of doses of I-BET151 and alisertib for 48 hours, as described in the methods, and images were obtained to quantify percentage confluence per well. Fraction affected (Fa) is defined as 100 − percent confluence and plotted on the x-axis. Each dose combination and the resultant Fa, as well as the Fa for each drug alone at individual doses, were entered into the Compusyn software. From these data, the CI for each dose combination was calculated, determined if the Fa observed was antagonistic (less than each drug's effect alone, CI >1), additive (equal to the effective of each drug alone, CI = 1), or greater-than additive (greater than the effect of each drug alone, CI <1). CI <0.7 is generally considered synergistic. For each cell line, a majority of drug combinations had CI <1, with Fa <0.5 for all such combinations. Representative experiments shown.
Figure 2
Figure 2
AURKA inhibition with alisertib induces increased RNA expression of oncogenes, which is mitigated by BRD4 inhibition with I-BET151. Cells were treated with 1 μM I-BET151, 1 μM alisertib, both drugs, or vehicle control for 24 hours, after which RNA was extracted from each cell line and used for RT-qPCR. Treatment with alisertib alone (dark gray bars) induced overexpression of most oncogenic targets tested in NB-SD (A), NB1643 (B) and SK-N-SH (C) cells, with less of an effect on SK-N-AS (D) cells except for AURKA. Co-treatment of the cells with I-BET151 reduced or entirely abrogated this overexpression for most target genes in all four cell lines. Relative expression shown using ddCt methods, with each gene's expression normalized first to housekeeping genes in each sample then against each gene's expression in the vehicle control. Three independent experiments performed, with mean and standard error plotted; one-way ANOVA based on treatment for NB-SD among the four treatment groups P = 0316, for NB1643 P = .0169, for SK-N-SH P = .0482, and for SK-N-AS P = .0079.
Figure 3
Figure 3
Dual AURKA and BRD4 inhibition is most efficacious in repressing target oncoprotein expression. Western blots of oncoprotein expression in four neuroblastoma cell lines. Cells were treated with 1 μM I-BET151, 1 μM alisertib, both drugs, or vehicle alone for 48 hours, after which cells were lysed and total protein harvested for Western blot. For all four cell lines, treatment with the AURKA inhibitor alisertib caused decreased p-AURKA levels and increased total AURKA expression, but with decreases in MYC and/or MYCN expression. Treatment with BRD4 inhibitor I-BET151 caused decreased in AURKA, MYC, and MYCN expression. Use of both inhibitors caused greater decrease in protein expression of AURKA, MYCN, and MYC as compared to alisertib alone in all three cell lines. Similar changes were seen on CDK4/6 and MCL1 in NB1643 and SK-N-SH cells and in CDK4 and BCL2 in NB-SD cells. SK-N-AS cells showed no effects on CDK6, MCL1, or BCL2 expression with any drug treatments. Immunoblot band intensity was quantified by densitometry and normalized against the actin control for each lane, then against the vehicle control for each cell line. Normalized expression for each band is shown directly below it. Three individual experiments were performed, with representative blots shown.
Figure 4
Figure 4
Drug combinations of I-BET151 and alisertib improve survival of mice with neuroblastoma tumor xenografts. Kaplan-Meier survival curves of tumor xenograft studies. See main text for description of treatment methods; n = 5 mice per treatment group. For mice treated with I-BET151, alisertib, both drugs, or vehicle alone, the two-drug combination was most efficacious as compared to vehicle control in extending survival (see main text for individual comparisons and P values, calculated by Mantel-Cox log-rank test).
Figure 5
Figure 5
Drug combinations of I-BET151 and alisertib with vincristine improve survival of mice with neuroblastoma tumor xenografts. Kaplan-Meier survival curves of tumor xenograft studies. See main text for description of treatment methods; n = 5 mice per treatment group. For mice treated with various combinations of I-BET151, alisertib, vincristine, and vehicle, the three-drug combination was most efficacious as compared to vehicle in extending survival (see main text for individual comparisons and P values, calculated by Mantel-Cox log-rank test).

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