Purpose KIT is the major oncogenic driver of gastrointestinal stromal tumors (GISTs). Imatinib, sunitinib and regorafenib are approved therapies; however, efficacy is often limited by the acquisition of polyclonal secondary resistance mutations in KIT, with those located in the activation (A) loop (exons 17/18) being particularly problematic. Here we explored the KIT inhibitory activity of ponatinib in preclinical models and describe initial characterization of its activity in GIST patients. Experimental Design The cellular and in vivo activities of ponatinib, imatinib, sunitinib and regorafenib against mutant KIT were evaluated using an accelerated mutagenesis assay and a panel of engineered and GIST-derived cell lines. The ponatinib-KIT co-structure was also determined. The clinical activity of ponatinib was examined in three GIST patients previously treated with all 3 FDA-approved agents. Results In engineered and GIST-derived cell lines, ponatinib potently inhibited KIT exon 11 primary mutants and a range of secondary mutants, including those within the A-loop. Ponatinib also induced regression in engineered and GIST-derived tumor models containing these secondary mutations. In a mutagenesis screen, 40 nM ponatinib was sufficient to suppress outgrowth of all secondary mutants except V654A, which was suppressed at 80 nM. This inhibitory profile could be rationalized based on structural analyses. Ponatinib (30 mg daily) displayed encouraging clinical activity in two of three GIST patients. Conclusion Ponatinib possesses potent activity against most major clinically-relevant KIT mutants, and has demonstrated preliminary evidence of activity in patients with refractory GIST. These data strongly support further evaluation of ponatinib in GIST patients.

Rhythmic mRNA expression is a hallmark of circadian biology and has been described in numerous experimental systems including mammals. A small number of core clock gene mRNAs and a much larger number of output mRNAs are under circadian control. The rhythmic expression of core clock genes is regulated at the transcriptional level, and this regulation is important for the timekeeping mechanism. However, the relative contribution of transcriptional and posttranscriptional regulation to global circadian mRNA oscillations is unknown. To address this issue in Drosophila, we isolated nascent RNA from adult fly heads collected at different time points and subjected it to high-throughput sequencing. mRNA was isolated and sequenced in parallel. Some genes had cycling nascent RNAs with no cycling mRNA, caused, most likely, by light-mediated read-through transcription. Most genes with cycling mRNAs had significant nascent RNA cycling amplitudes, indicating a prominent role for circadian transcriptional regulation. However, a considerable fraction had higher mRNA amplitudes than nascent RNA amplitudes. The same comparison for core clock gene mRNAs gives rise to a qualitatively similar conclusion. The data therefore indicate a significant quantitative contribution of posttranscriptional regulation to mRNA cycling.Nas-seq | chromatin dynamics T he circadian clock controls the rhythmic expression of thousands of genes. These mRNAs mediate the oscillation of numerous biochemical, physiological, and behavioral functions. Although inconsistency among microarray datasets has made it difficult to identify a definitive set of mRNA cyclers in Drosophila, there is good agreement on a small number of robust cycling mRNAs (see below). Moreover, it appears that a much larger number of cycling mRNAs exist within circadian neurons (1), and thousands of mRNAs have been shown to oscillate within individual tissues in mammals (2-5).Cycling mRNAs are dependent on a functional pacemaker (6, 7), but most probably are regulated only indirectly by the core circadian clock (8). In flies, this mechanism consists of the CLOCK and CYCLE heterodimer (CLK/CYC), which drives the rhythmic transcription of its repressor protein genes period and timeless (9). After translation, the proteins PER and TIM decrease their own transcription by inhibiting the activity of CLK/ CYC (10-12). A set of largely orthologous proteins (CLK, BMAL1, PER, and CRYPTOCHROME) functions similarly in mammals. Although posttranslational regulation of these transcription factors makes a major contribution to core clock function (13-19), there is evidence that transcriptional regulation also is important for circadian timing. For example, flies with enhanced CLK/CYC-mediated transcription have unusually high levels of per mRNA and significantly shorter periods (20).The core clock model drives the assumption that the large population of cycling mRNAs-the hundreds or thousands of output mRNAs under circadian regulation-is also under transcriptional regulation. However, it is not known w...

We sequenced Drosophila head RNA to identify a small set of miRNAs that undergo robust circadian cycling. We concentrated on a cluster of six miRNAs, mir-959-964, all of which peak at about ZT12 or lights-off. The cluster pri-miRNA is transcribed under bona fide circadian transcriptional control, and all 6 mature miRNAs have short half-lives, a requirement for cycling. A viable Gal4 knock-in strain localizes prominent cluster miRNA expression to the adult head fat body. Analysis of cluster knock-out and over-expression strains indicates that innate immunity, metabolism, and feeding behavior are under cluster miRNA regulation. Manipulation of food intake also affects the levels and timing of cluster miRNA transcription with no more than minor effects on the core circadian oscillator. These observations indicate a feedback circuit between feeding time and cluster miRNA expression-function as well as a surprising role of post-transcriptional regulation in the circadian control of these phenotypes.