Mdm2 is the major negative regulator of p53 tumor-suppressor activity. This oncoprotein is overexpressed in many human tumors that retain the wild-type p53 allele. As such, targeted inhibition of Mdm2 is being considered as a therapeutic anticancer strategy. The N-terminal hydrophobic pocket of Mdm2 binds to p53 and thereby inhibits the transcription of p53 target genes. Additionally, the C-terminus of Mdm2 contains a RING domain with intrinsic ubiquitin E3 ligase activity. By recruiting E2 ubiquitin-conjugating enzyme(s), Mdm2 acts as a molecular scaffold to facilitate p53 ubiquitination and proteasome-dependent degradation. Mdmx (Mdm4), an Mdm2 homolog, also has a RING domain and hetero-oligomerizes with Mdm2 to stimulate its E3 ligase activity. Recent studies have shown that C-terminal residues adjacent to the RING domain of both Mdm2 and Mdmx contribute to Mdm2 E3 ligase activity. However, the molecular mechanisms mediating this process remain unclear, and the biological consequences of inhibiting Mdm2/Mdmx co-operation or blocking Mdm2 ligase function are relatively unexplored. This study presents biochemical and cell biological data that further elucidate the mechanisms by which Mdm2 and Mdmx co-operate to regulate p53 level and activity. We use chemical and genetic approaches to demonstrate that functional inhibition of Mdm2 ubiquitin ligase activity is insufficient for p53 activation. This unexpected result suggests that concomitant treatment with Mdm2/Mdmx antagonists may be needed to achieve therapeutic benefit.

REV-ERBα and REV-ERBβ nuclear receptors regulate several physiological processes, including circadian rhythm and metabolism. A previous study reported the REV-ERBα gene to be co-overexpressed with ERBB2 in breast cancer cell lines. Surprisingly, we found that several tumor types, including a number of breast cancer cell lines, predominantly express the REV-ERBβ variant. This pattern was independent of ERBB2 and ER status, and opposite to that of non-cancer mammary epithelial HMEC cells, in which REV-ERBα was the major variant. Consistent with this molecular profile, REV-ERB target genes in both circadian and metabolic pathways were derepressed upon silencing of REV-ERBβ, but not REV-ERBα. Strikingly, we found that REV-ERBβ is a determinant of sensitivity to chloroquine, a clinically relevant lysosomotropic agent that suppresses autophagy. The cytoprotective function of REV-ERBβ appears to operate downstream of autophagy blockade. Through compound screening, we identified ARN5187, a novel lysosomotropic REV-ERBβ ligand with a dual inhibitory activity toward REV-ERB-mediated transcriptional regulation and autophagy. Remarkably, although ARN5187 and chloroquine share similar lysosomotropic potency and have a similar effect on autophagy inhibition, ARN5187 is significantly more cytotoxic. Collectively, our results reveal that dual inhibition of REV-ERBβ and autophagy is an effective strategy for eliciting cytotoxicity in cancer cells. Furthermore, our discovery of a novel inhibitor compound of both REV-ERB and autophagy may provide a scaffold for the discovery of new multifunctional anticancer agents.

Pediatric community-acquired pneumonia (CAP) is often treated with 10 days of antibiotics. Shorter treatment strategies may be effective and lead to less resistance. The impact of duration of treatment on the respiratory microbiome is unknown. Data are from children (n = 171), ages 6 to 71 months, enrolled in the SCOUT-CAP trial (NCT02891915). Children with CAP were randomized to a short (5 days) versus standard (10 days) beta-lactam treatment strategy. Throat swabs were collected at enrollment and the end of the study and used for shotgun metagenomic sequencing. The number of beta-lactam and multidrug efflux resistance genes per prokaryotic cell (RGPC) was significantly lower in children receiving the short compared to standard treatment strategy at the end of the study (Wilcoxon rank sum test, P < 0.05 for each). Wilcoxon effect sizes were small for beta-lactam (r: 0.15; 95% confidence interval [CI], 0.01 to 0.29) and medium for multidrug efflux RGPC (r: 0.23; 95% CI, 0.09 to 0.37). Analyses comparing the resistome at the beginning and end of the trial indicated that in contrast to the standard strategy group, the resistome significantly differed in children receiving the short course strategy. Relative abundances of commensals such as Neisseria subflava were higher in children receiving the standard strategy, and Prevotella species and Veillonella parvula were higher in children receiving the short course strategy. We conclude that children receiving 5 days of beta-lactam therapy for CAP had a significantly lower abundance of antibiotic resistance determinants than those receiving standard 10-day treatment. These data provide an additional rationale for reductions in antibiotic use when feasible. IMPORTANCE Antibiotic resistance is a major threat to public health. Treatment strategies involving shorter antibiotic courses have been proposed as a strategy to lower the potential for antibiotic resistance. We examined relationships between the duration of antibiotic treatment and its impact on resistance genes and bacteria in the respiratory microbiome using data from a randomized controlled trial of beta-lactam therapy for pediatric pneumonia. The randomized design provides reliable evidence of the effectiveness of interventions and minimizes the potential for confounding. Children receiving 5 days of therapy for pneumonia had a lower prevalence of two different types of resistance genes than did those receiving the 10-day treatment. Our data also suggest that children receiving longer durations of therapy have a greater abundance of antibiotic resistance genes for a longer period of time than do children receiving shorter durations of therapy. These data provide an additional rationale for reductions in antibiotic use.