Aurora A Kinase Inhibition Is Synthetic Lethal with Loss of the RB1 Tumor Suppressor Gene.

Loss-of-function mutations in the retinoblastoma gene RB1 are common in several treatment-refractory cancers such as small-cell lung cancer and triple-negative breast cancer. To identify drugs synthetic lethal with RB1 mutation (RB1 mut), we tested 36 cell-cycle inhibitors using a cancer cell panel profiling approach optimized to discern cytotoxic from cytostatic effects. Inhibitors of the Aurora kinases AURKA and AURKB showed the strongest RB1 association in this assay. LY3295668, an AURKA inhibitor with over 1,000-fold selectivity versus AURKB, is distinguished by minimal toxicity to bone marrow cells at concentrations active against RB1 mut cancer cells and leads to durable regression of RB1 mut tumor xenografts at exposures that are well tolerated in rodents. Genetic suppression screens identified enforcers of the spindle-assembly checkpoint (SAC) as essential for LY3295668 cytotoxicity in RB1-deficient cancers and suggest a model in which a primed SAC creates a unique dependency on AURKA for mitotic exit and survival. SIGNIFICANCE: The identification of a synthetic lethal interaction between RB1 and AURKA inhibition, and the discovery of a drug that can be dosed continuously to achieve uninterrupted inhibition of AURKA kinase activity without myelosuppression, suggest a new approach for the treatment of RB1-deficient malignancies, including patients progressing on CDK4/6 inhibitors.See related commentary by Dick and Li, p. 169.This article is highlighted in the In This Issue feature, p. 151.

Alternative splicing within the ligand binding domain of the human constitutive androstane receptor.

The human constitutive androstane receptor (hCAR; NR1I3) is a member of the nuclear receptor superfamily. The activity of hCAR is regulated by a variety of xenobiotics including clotrimazole and acetaminophen metabolites. hCAR, in turn, regulates a number of genes responsible for xenobiotic metabolism and transport including several cytochrome P450s (CYP 2B5, 2C9, and 3A4) and the multidrug resistance-associated protein 2 (MRP2, ABCC2). Thus, hCAR is believed to be a mediator of drug-drug interactions. We identified two novel hCAR splice variants: hCAR2 encodes a receptor in which alternative splice acceptor sites are utilized resulting in a 4 amino acid insert between exons 6 and 7, and a 5 amino acid insert between 7 and 8, and hCAR3 encodes a receptor with exon 7 completely deleted resulting in a 39 amino acid deletion. Both hCAR2 and hCAR3 mRNAs are expressed in a pattern similar to the initially described MB67 (hCAR1) with some key distinctions. Although the levels of expression vary depending on the tissue examined, hCAR2 and hCAR3 contribute 6-8% of total hCAR mRNA in liver. Analysis of the activity of these variants indicates that both hCAR2 and hCAR3 lose the ability to heterodimerize with RXR and lack transactivation activity in cotransfection experiments where either full-length receptor or GAL4 DNA-binding domain/CAR ligand binding domain chimeras were utilized. Although the role of hCAR2 and hCAR3 is currently unclear, these additional splice variants may provide for increased diversity in terms of responsiveness to xenobiotics.

Liver X receptor and retinoic X receptor mediated ABCA1 regulation and cholesterol efflux in macrophage cells-messenger RNA measured by branched DNA technology.

ABCA1 is an ATP binding cassette transporter that plays an essential role in cholesterol and phospholipid efflux and HDL biogenesis. ABCA1 expression in macrophage cells is subject to regulation by cAMP, cholesterol loading, and ligands of the nuclear receptors liver X receptor (LXR) and retinoid X receptor (RXR). We report here the development of a rapid and high volume branched DNA (bDNA) method to measure ABCA1 mRNA. By using the bDNA method, we show that both LXR and RXR ligands effectively regulate ABCA1 expression in three macrophage cell types: mouse RAW264.7 cell line, mouse peritoneal macrophage cells, and human macrophage THP-1 cells and their regulation is additive. Furthermore, by using a radiolabeled cholesterol efflux assay, we show that both LXR and RXR ligands are sufficient to mediate cholesterol efflux in macrophage cells and their efficacy correlates with ABCA1 regulation. These studies strengthen further the notion that LXR and RXR mediate ABCA1 expression and cholesterol efflux in macrophage cells as a permissive heterodimer and development of small molecule ligands of these nuclear receptors may represent a promising approach to modulating cholesterol efflux and plasma HDL cholesterol level in humans.