SUMMARY Accelerating cures for children with cancer remains an immediate challenge as a result of extensive oncogenic heterogeneity between and within histologies, distinct molecular mechanisms evolving between diagnosis and relapsed disease, and limited therapeutic options. To systematically prioritize and rationally test novel agents in preclinical murine models, researchers within the Pediatric Preclinical Testing Consortium are continuously developing patient-derived xenografts (PDXs)—many of which are refractory to current standard-of-care treatments—from high-risk childhood cancers. Here, we genomically characterize 261 PDX models from 37 unique pediatric cancers; demonstrate faithful recapitulation of histologies and subtypes; and refine our understanding of relapsed disease. In addition, we use expression signatures to classify tumors for TP53 and NF1 pathway inactivation. We anticipate that these data will serve as a resource for pediatric oncology drug development and will guide rational clinical trial design for children with cancer.

The VapBC toxin-antitoxin (TA) family is the largest of nine identified TA families. The toxin, VapC, is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. Although the VapBCs are the largest TA family, little is known about their biological roles. Here we describe a new general method for the overexpression and purification of toxic VapC proteins and subsequent determination of their RNase sequence-specificity. Functional VapC was isolated by expression of the nontoxic VapBC complex, followed by removal of the labile antitoxin (VapB) using limited trypsin digestion. We have then developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This technique employs the use of Pentaprobes as substrates for VapC. These are RNA sequences encoding every combination of five bases. We combine the RNase reaction with MALDI-TOF MS to detect and analyze the cleavage products and thus determine the RNA cut sites. Successful MALDI-TOF MS analysis of RNA fragments is acutely dependent on sample preparation methods. The sequencespecificity of four VapC proteins from two different organisms (VapC PAE0151 and VapC PAE2754 from Pyrobaculum aerophilum, and VapC Rv0065 and VapC Rv0617 from Mycobacterium tuberculosis) was successfully determined using the described strategy. This rapid and sensitive method can be applied to determine the sequence-specificity of VapC ribonucleases along with other RNA interferases (such as MazF) from a range of organisms.

In recent years, evidence has emerged for the existence of many diverse types of RNA, which play roles in a wide range of biological processes in all kingdoms of life. These molecules generally do not, however, act in isolation, and identifying which proteins partner with RNA is a major challenge. Many methods, in vivo and in vitro, have been used to address this question, including combinatorial or high-throughput approaches, such as systematic evolution of ligands, cross-linking and immunoprecipitation and RNA immunoprecipitation combined with deep sequencing. However, most of these methods are not trivial to pursue and often require substantial optimization before results can be achieved. Here, we demonstrate a simple technique that allows one to screen proteins for RNA-binding properties in a gel-shift experiment and can be easily implemented in any laboratory. This assay should be a useful first-pass tool for assessing whether a protein has RNA- or DNA-binding properties, prior to committing resources to more complex procedures.

␣-Hemoglobin (␣Hb) stabilizing protein (AHSP) is expressed in erythropoietic tissues as an accessory factor in hemoglobin synthesis. AHSP forms a specific complex with ␣Hb and suppresses the heme-catalyzed evolution of reactive oxygen species by converting ␣Hb to a conformation in which the heme is coordinated at both axial positions by histidine side chains (bis-histidyl coordination). Currently, the detailed mechanism by which AHSP induces structural changes in ␣Hb has not been determined. Here, we present x-ray crystallography, NMR spectroscopy, and mutagenesis data that identify, for the first time, the importance of an evolutionarily conserved proline, Pro 30 , in loop 1 of AHSP. Mutation of Pro 30 to a variety of residue types results in reduced ability to convert ␣Hb. In complex with ␣Hb, AHSP Pro 30 adopts a cis-peptidyl conformation and makes contact with the N terminus of helix G in ␣Hb. Mutations that stabilize the cis-peptidyl conformation of free AHSP, also enhance the ␣Hb conversion activity. These findings suggest that AHSP loop 1 can transmit structural changes to the heme pocket of ␣Hb, and, more generally, highlight the importance of cis-peptidyl prolyl residues in defining the conformation of regulatory protein loops.Mammalian adult hemoglobin (HbA) 5 is a tetramer of two ␣Hb and two ␤Hb subunits, which is produced to extremely high concentrations (ϳ340 mg/ml) in red blood cells. Numerous mechanisms exist to balance and coordinate HbA synthesis in normal erythropoiesis, and problems with the production of either HbA subunit give rise to thalassemia, a common cause of anemia worldwide. Previously, we identified ␣-hemoglobin stabilizing protein (AHSP) as an accessory factor in normal HbA production (1). AHSP forms a dimeric complex with ␣Hb (see Fig. 1A) (2) but does not interact with ␤Hb or HbA. AHSP also binds heme-free (apo) ␣Hb (3) and may serve functions in both the folding of nascent ␣Hb (4) and the detoxification of excess ␣Hb that remains following HbA assembly (2, 5). Mice carrying an Ahsp gene knock-out display mild anemia, ineffective erythropoiesis, and enhanced sensitivity to oxidative stress (1, 6), features also observed in ␤-thalassemia patients due to the cytotoxic effects of free ␣Hb.Free ␣Hb promotes the formation of harmful reactive oxygen species as a result of reduction/oxidation reactions involving the heme iron (7,8). Reactive oxygen species can damage heme, ␣Hb, and other cellular structures, resulting in hemoglobin precipitates and death of erythroid precursor cells (9 -12). The presence of AHSP may explain how cells tolerate the slight excess of ␣Hb that is observed in normal erythropoiesis, which is postulated to inhibit the formation of non-functional ␤Hb tetramers, thus providing a robust mechanism for achieving the correct subunit stoichiometry during HbA assembly (13).Structural and biochemical studies have begun to elucidate the molecular mechanism by which AHSP detoxifies ␣Hb. AHSP binds to oxygenated ␣Hb to generate an initial complex that retains the oxy-hem...