Replication Study: BET bromodomain inhibition as a therapeutic strategy to target c-Myc

Abstract

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Kandela et al., 2015) that described how we intended to replicate selected experiments from the paper "BET bromodomain inhibition as a therapeutic strategy to target c-Myc" (Delmore et al., 2011). Here we report the results of those experiments. We found that treatment of human multiple myeloma (MM) cells with the small-molecular inhibitor of BET bromodomains, (+)-JQ1, selectively downregulated MYC transcription, which is similar to what was reported in the original study (Figure 3B; Delmore et al., 2011). Efficacy of (+)-JQ1 was evaluated in an orthotopically xenografted model of MM. Overall survival was increased in (+)-JQ1 treated mice compared to vehicle control, similar to the original study (Figure 7E; Delmore et al., 2011). Tumor burden, as determined by bioluminescence, was decreased in (+)-JQ1 treated mice compared to vehicle control; however, while the effect was in the same direction as the original study (Figure 7C-D; Delmore et al., 2011), it was not statistically significant. The opportunity to detect a statistically significant difference was limited though, due to the higher rate of early death in the control group, and increased overall survival in (+)-JQ1 treated mice before the pre-specified tumor burden analysis endpoint. Additionally, we evaluated the (-)-JQ1 enantiomer that is structurally incapable of inhibiting BET bromodomains, which resulted in a minimal impact on MYC transcription, but did not result in a statistically significant difference in tumor burden or survival distributions compared to treatment with (+)-JQ1. Finally, we report meta-analyses for each result.

Keywords: Reproducibility Project: Cancer Biology; bromodomain inhibitor; cancer biology; human; metascience; mouse; myeloma; replication; reproducibility.

Figure 1.. MYC expression in JQ1-treated MM.1S-luc cells.

MM.1S-luc cells were treated with 500 nM (+)-JQ1, 500 nM (−)-JQ1, or an equivalent volume of DMSO. Total RNA was isolated at 0 hr, 1 hr, and 8 hr after treatment and qRT-PCR analysis was performed to detect MYC and GAPDH levels. Relative expression (MYC/GAPDH) is presented for each time point and condition normalized to (+)-JQ1 treated cells at 0 hr. Means reported and error bars represent s.d. from five independent biological repeats. Mixed-design analysis of variance (ANOVA) with time (0 hr, 1 hr, and 8 hr) as the within-subjects factor and treatment ((+)-JQ1, (−)-JQ1, or vehicle) as the between-subjects factor; interaction effect: F(4,24) = 268.9, p=1.49x10⁻¹⁹, treatment main effect: F(2,12) = 393.5, p=1.15x10⁻¹¹, time main effect: F(2, 24) = 368.0, p=9.84x10⁻¹⁹. Planned paired t-test of MM.1S-luc cells harvested 8 hr after (+)-JQ1 treatment compared to cells 0 hr after (+)-JQ1 treatment; t(4) = 38.92, uncorrected p=2.60x10⁻⁶, a priori Bonferroni adjusted significance threshold = 0.025; (Bonferroni corrected p=5.21x10⁻⁶). Planned paired t-test of MM.1S-luc cells harvested 1 hr after (+)-JQ1 treatment compared to cells 0 hr after (+)-JQ1 treatment; t(4) = 25.10, uncorrected p=1.50x10⁻⁵, a priori Bonferroni adjusted significance threshold = 0.025; (Bonferroni corrected p=2.99x10⁻⁵). Additional details for this experiment can be found at https://osf.io/9swnx/. DOI: http://dx.doi.org/10.7554/eLife.21253.002

Figure 2.. Overall survival in JQ1-treated MM.1S-luc orthotopic xenograft model.

Kaplan-Meier plot of overall survival during the pre-specified study period of 36 days. Female SCID-beige mice with established MM.1S-luc orthotopic xenografts were randomized to daily IP injections of 50 mg/kg (+)-JQ1, 50 mg/kg (−)-JQ1, or vehicle control. Number of mice: n = 12 for each group. Log-rank (Mantel-Cox) test of (+)-JQ1 treatment compared to vehicle control; uncorrected p=0.024, a priori Bonferroni adjusted alpha level = 0.025; (Bonferroni corrected p=0.047). Log-rank (Mantel-Cox) test of (+)-JQ1 compared to (−)-JQ1; uncorrected p=0.093, a priori Bonferroni adjusted alpha level = 0.025; (Bonferroni corrected p=0.187). Additional details for this experiment can be found at https://osf.io/pnvtd/. DOI: http://dx.doi.org/10.7554/eLife.21253.003

Figure 3.. Tumor burden in JQ1-treated MM.1S-luc orthotopic xenograft model.

Female SCID-beige mice were orthotopically xenografted after intravenous injection with MM.1S-luc cells. Following detection of established disease (diffusely engrafted in the skeleton with an increase in bioluminescence), mice were randomly assigned to receive daily IP injections of 50 mg/kg (+)-JQ1, 50 mg/kg (−)-JQ1, or vehicle control (VEH). (A) Representative whole-body bioluminescence images of mice bearing MM.1S tumors 22 days after the start of the indicated treatment. (B) Line graph of tumor burden, as measured by whole-body bioluminescence, of tumor bearing mice during the course of the indicated treatment. Means reported and error bars represent s.e.m. The number of mice per condition at start of treatment (n = 12 for each group) and at day 22 ((+)-JQ1 = 11, (−)-JQ1 = 9, Vehicle = 7). One-way ANOVA on a natural log transformed day 22 bioluminescence signal; F(2,24) = 1.126, p=0.341. The pairwise contrast between (+)-JQ1 treatment and Vehicle; Fisher’s LSD test; t(24) = 1.303, p=0.205 with a priori alpha level = 0.05. The pairwise contrast between (+)-JQ1 and (−)-JQ1 treatment; Fisher’s LSD test; t(24) = 1.221, p=0.234 with a priori alpha level = 0.05. Additional details for this experiment can be found at https://osf.io/pnvtd/. DOI: http://dx.doi.org/10.7554/eLife.21253.004

Figure 3—figure supplement 1.. Individual tumor xenografts.

This is the same experiment as in Figure 3, but with the bioluminescence data plotted on a continuous scale (A) or natural log transformed scale (B) for each animal rather than averages. Female SCID-beige mice with established MM.1S-luc orthotopic xenografts were randomly assigned to receive daily IP injections of 50 mg/kg (+)-JQ1, (−)-JQ1, or vehicle control. The number of mice per condition at start of treatment (n = 12 for each group) and at day 22 ((+)-JQ1 = 11, (−)-JQ1 = 9, Vehicle = 7). Additional details for this experiment can be found at https://osf.io/pnvtd/. DOI: http://dx.doi.org/10.7554/eLife.21253.005

Figure 4.. Meta-analyses of each effect.

DOI: http://dx.doi.org/10.7554/eLife.21253.006