Domain-selective BET inhibition attenuates transcriptional and behavioral responses to cocaine

Abstract

Epigenetic pharmacotherapies have emerged as a promising treatment option for substance use disorder (SUD) due to their ability to reverse maladaptive transcriptional and behavioral responses to drugs of abuse. In particular, inhibitors of bromodomain and extra terminal domain (BET) reader proteins have been shown to reduce cocaine- and opioid-seeking behaviors in rodents. However, only pan-BET inhibitors, small molecules that bind to both bromodomains (BD1 and BD2) with all BET proteins, have been investigated in animal models of SUD. Given the potential side effects associated with pan-BET inhibitors, safer and more selective strategies are needed to advance BET therapeutics as a potential treatment for SUD. Here, we show that RVX-208, a clinically tested, BD2-selective BET inhibitor, dose-dependently reduced cocaine conditioned place preference in male and female mice, similar to the pan-BET inhibitor JQ1. In other behavioral experiments, RVX-208 treatment did not alter distance traveled, anxiety-like behavior, or novel object recognition memory. At the transcriptional level, RVX-208 attenuated the expression of multiple cocaine-induced genes in the nucleus accumbens in a sex-dependent manner. RVX-208 produced a distinct transcriptional response in stimulated primary neurons compared to JQ1 but had little effect on gene expression in non-stimulated neurons. Together, these data indicate that targeting domain-specific BET mechanisms may be an effective and safer strategy to reduce cocaine-induced neurobehavioral adaptations.

Keywords: Addiction; BET; BRD4; Bromodomain; Cocaine; Epigenetics; Substance use disorder.

Figure 1:. RVX-208 has limited effects in non-stimulated primary neurons.

Primary neurons were treated with vehicle (n = 9), RVX-208 (n = 6), or JQ1 (n = 6) (100 nM and 500 nM) for 1, 2, and 3 hours. Following treatment, (A) Bdnf, (B) Arc, (C) Nr4a1, (D) c-fos, and (E) Gria1 expression levels were measured via RT-qPCR. Using Bonferroni post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate a significant difference from vehicle at the same timepoint. Data are mean ±SEM.

Figure 2:. RVX-208 alters the expression of multiple genes in BDNF stimulated primary neurons.

Primary neurons were treated with BDNF, vehicle + BDNF, RVX-208 + BDNF or JQ1 + BDNF for 2 hours. The expression of (A) Bdnf, (B) Arc, (C) Nr4a1, (D) c-fos, and (E) Gria1 was measured via RT-qPCR. Horizontal dotted line indicates baseline expression of untreated neurons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate a significant difference compared to BDNF or Vehicle + BDNF using Bonferroni post hoc test. Data are mean ±SEM, n = 6 per treatment.

Figure 3:. RVX-208 has no effect on gene expression in AMPA stimulated primary neurons.

Primary neurons were treated with AMPA, vehicle + AMPA, RVX-208 + AMPA, or JQ1 + AMPA for 2 hours. The expression of (A) Bdnf, (B) Arc, (C) Nr4a1, (D) c-fos, and (E) Gria1 was measured via RT-qPCR. Horizontal dotted line indicates baseline expression of untreated neurons. *P < 0.05 and ****P < 0.0001 indicate a significant difference compared to AMPA or Vehicle + AMPA using Bonferroni post hoc test. Data are mean ±SEM, n = 6 per treatment.

Figure 4:. RVX-208 alters cocaine-induced gene expression in the NAc.

Male and female mice were treated with vehicle/saline, vehicle/cocaine, RVX-208/saline, or RVX-208/cocaine. Following treatment, (A) Bdnf, (B) Arc (C), Nr4a1 (D), c-fos, (E) Fosb, and (F) Gria1 expression levels were measured via RT-qPCR. *P < 0.05, ***P < 0.001, and ****P < 0.0001 indicate a significant difference using Bonferroni post hoc test. Data are mean ±SEM, n = 8 per treatment.

Figure 5.. Effects of RVX-208 on behavior.

(A) Dose-dependent reduction of cocaine conditioned place preference (CPP) by RVX-208 compared to vehicle in male and female mice (n = 8–13). (B) The effects of RVX-208 (50 mg/kg) on locomotor activity and (C) time spent in the inner and outer zones in open field in male and female mice (n = 8). (D) Time spent exploring objects during novel object recognition (NOR) pretest in male and female mice (n = 10). (E) Time spent exploring the novel object (NO) and familiar object (FO) during the NOR test in vehicle and RVX-208 (50 mg/kg) treated male and female mice (n = 10). (F) NOR discrimination index (DI) values for vehicle and RVX-208 treated male and female mice (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001 indicate a significant difference using Bonferroni post hoc test. Data are mean ±SEM.