BACKGROUND AND PURPOSEThe cyclin-dependent kinase CDK9 is an important therapeutic target but currently available inhibitors exhibit low specificity and/or narrow therapeutic windows. Here we have used a new highly specific CDK9 inhibitor, LDC000067 to interrogate gene control mechanisms mediated by CDK9. EXPERIMENTAL APPROACHThe selectivity of LDC000067 was established in functional kinase assays. Functions of CDK9 in gene expression were assessed with in vitro transcription experiments, single gene analyses and genome-wide expression profiling. Cultures of mouse embryonic stem cells, HeLa cells, several cancer cell lines, along with cells from patients with acute myelogenous leukaemia were also used to investigate cellular responses to LDC000067. KEY RESULTSThe selectivity of LDC000067 for CDK9 over other CDKs exceeded that of the known inhibitors flavopiridol and DRB. LDC000067 inhibited in vitro transcription in an ATP-competitive and dose-dependent manner. Gene expression profiling of cells treated with LDC000067 demonstrated a selective reduction of short-lived mRNAs, including important regulators of proliferation and apoptosis. Analysis of de novo RNA synthesis suggested a wide ranging positive role of CDK9. At the molecular and cellular level, LDC000067 reproduced effects characteristic of CDK9 inhibition such as enhanced pausing of RNA polymerase II on genes and, most importantly, induction of apoptosis in cancer cells. CONCLUSIONS AND IMPLICATIONSOur study provides a framework for the mechanistic understanding of cellular responses to CDK9 inhibition. LDC000067 represents a promising lead for the development of clinically useful, highly specific CDK9 inhibitors. AbbreviationsLDC067, LDC000067, 3-((6-(2-methoxyphenyl)pyrimidin-4-yl)amino)phenyl) methane sulfonamide; AML, acute myelogenous leukaemia; CDK, cyclin-dependent kinase; ChIP, chromatin immunoprecipitation; CTD, carboxyterminal domain; DRB, 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole; DSIF, DRB sensitivity-inducing factor; FRET, fluorescence resonance energy transfer; mESCs, mouse embryonic stem cells; NELF, negative elongation factor; P-TEFb, positive transcription elongation factor b; RNAPII, RNA polymerase II; RT-qPCR, reverse transcription-quantitative real-time PCR; Ser2/5/7-P, CTD phospho-Ser 2/5/7

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The hypothalamus plays a crucial role in the control of the energy balance and also retains neurogenic potential into adulthood. Recent studies have reported the severe alteration of the cell turn-over in the hypothalamus of obese animals and it has been proposed that a neurogenic deficiency in the hypothalamus could be involved in the development of obesity. To explore this possibility, we examined hypothalamic cell renewal during the homeostatic response to dietary fat in mice, i.e., at the onset of diet-induced obesity. We found that switching to high-fat diet (HFD) accelerated cell renewal in the hypothalamus through a local, rapid and transient increase in cell proliferation, peaking three days after introducing the HFD. Blocking HFD-induced cell proliferation by central delivery of an antimitotic drug prevented the food intake normalization observed after HFD introduction and accelerated the onset of obesity. This result showed that HFD-induced dividing brain cells supported an adaptive anorectic function. In addition, we found that the percentage of newly generated neurons adopting a POMC-phenotype in the arcuate nucleus was increased by HFD. This observation suggested that the maturation of neurons in feeding circuits was nutritionally regulated to adjust future energy intake. Taken together, these results showed that adult cerebral cell renewal was remarkably responsive to nutritional conditions. This constituted a physiological trait required to prevent severe weight gain under HFD. Hence this report highlighted the amazing plasticity of feeding circuits and brought new insights into our understanding of the nutritional regulation of the energy balance.