No abstract

Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal, monocyte-derived dendritic cells or macrophages in Langerhans Cell (LCH) and non-Langerhans (non-LCH) histiocytoses, respectively. The discovery of BRAFV600E mutations in ~50% of these patients provided the first molecular therapeutisc target in histiocytosis. However, recurrent driving mutations in the majority of BRAFV600E-wildtype, non-LCH patients are unknown, and recurrent cooperating mutations in non-MAP kinase pathways are undefined for the histiocytic neoplasms. Through combined whole exome and transcriptome sequencing, we identified recurrent kinase fusions involving BRAF, ALK, and NTRK1, as well as recurrent, activating MAP2K1 and ARAF mutations in BRAFV600E-wildtype, non-LCH patients. In addition to MAP kinase pathway lesions, recurrently altered genes involving diverse cellular pathways were identified. Treatment of MAP2K1- and ARAF-mutated, non-LCH patients using MEK and RAF inhibitors, respectively, resulted in clinical efficacy demonstrating the importance of detecting and targeting diverse kinase alterations in these disorders.

Herein, we report the development of a microarray platform to select RNA motif-ligand interactions that allows simultaneous screening of both RNA and chemical space. We used this platform to identify the RNA internal loops that bind 6'- N-5-hexynoate kanamycin A ( 1). Selected internal loops that bind 1 were studied in detail and commonly display an adenine across from a cytosine independent of the size of the loop. Additional preferences are also observed. For 3 x 3 nucleotide loops, there is a preference for purines, and for 2 x 2 nucleotide loops there is a preference for pyrimidines neighbored by an adenine across from a cytosine. This technique has several advantageous features for selecting RNA motif-ligand interactions: (1) higher affinity RNA motif-ligand interactions are identified by harvesting bound RNAs from lower ligand loadings; (2) bound RNAs are harvested from the array via gel extraction, mitigating kinetic biases in selections; and (3) multiple selections are completed on a single array surface. To further demonstrate that multiple selections can be completed in parallel on the same array surface, we selected the RNA internal loops from a 4096-member RNA internal loop library that bound a four-member aminoglycoside library. These experiments probed 16,384 (4 aminoglycoside x 4096-member RNA library) interactions in a single experiment. These studies allow for parallel screening of both chemical and RNA space to improve our understanding of RNA-ligand interactions. This information may facilitate the rational and modular design of small molecules targeting RNA.

Herein, we report the RNA hairpin loops from a 6-nucleotide hairpin library that bind 6′-acylated kanamycin A (1) and 6′-acylated neamine (2) identified by 2-Dimensional Combinatorial Screening (2DCS). Hairpins selected to bind 1 have K d 's ranging from 235-1035 nM, with an average K d of 618 nM. For 2, the selected hairpins bind with K d 's ranging from 135-2300 nM, with an average K d of 1010 nM. The selected RNA hairpin-ligand interactions are also specific for the ligand that they were selected to bind compared to the other arrayed ligand. For example, the mixture of hairpins selected for 1 on average bind 33-fold more tightly to 1 than 2 while the mixtures of hairpins selected for 2 on average bind 11-fold more tightly to 2 than 1. Secondary structure prediction of the selected sequences was completed to determine the motifs that each ligand binds, and the hairpin loop preferences for 1 and 2 were computed. For 1, the preferred hairpin loops contain an adenine separated by at least two nucleotides from a cytosine, for example ANNCNN (two-tailed p-value = 0.0010) and ANNNCN (two-tailed p-value <0.0001). For 2, the preferred hairpin loops contain both 5′GC and 5′CG steps (two-tailed p-value <0.0001). These results expand the information available on the RNA hairpin loops that bind small molecules and could prove useful for targeting RNA.RNA plays important roles in biological systems beyond the transfer of genetic material. For example, microRNAs regulate RNA lifetime and contribute to cancer,(1) riboswitches control gene expression by interacting with metabolites,(2) and viral RNAs facilitate translation of viral proteins (3). The most studied RNA therapeutic target for small molecules is the bacterial ribosome; most anti-bacterials that target the ribosome form direct contacts with RNA.(4) Other RNAs have been targeted with small molecules including HIV Trans-Activating Response (TAR) RNA (5) and Rev-Responsive Element (RRE) . Despite these studies, most RNA drug targets represent untapped potential.One difficulty in exploiting other RNA targets for small molecule therapeutics is the relatively limited information available about the small RNA motifs that small molecules bind. What is known about RNA-binding ligands has come from studying smaller motifs or domains derived from RNA therapeutic targets. Such approaches have helped develop compounds to inhibit HIV infection by targeting TAR RNA,(7) to inhibit bacterial growth by targeting the bacterial *Author to whom correspondence should be addressed; Email: E-mail: mddisney@buffalo.edu Phone: (716) (9,10) and to facilitate the elimination of plasmids that cause antibiotic resistance (11,12).To develop rational approaches to target RNA, information on the ligands that like to bind RNA and the RNA motifs that like to bind ligands is needed. The most commonly used methods to gather such information are systematic evolution of ligands by exponential enrichment (SELEX) and high throughput screening. In SELEX, RNAs that bind a ligand of interest with hig...

Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. Here, we analyzed pediatric and adult pineoblastoma samples (n = 23) using a combination of genome-wide DNA methylation profiling and whole-exome sequencing or whole-genome sequencing. Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower-grade pineal tumors and normal pineal gland. Recurrent variants were found in genes involved in PKA- and NF-κB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expresion of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain.