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Background ABL-class fusions including NUP214-ABL1 and EBF1-PDGFRB occur in high risk acute lymphoblastic leukaemia (ALL) with gene expression patterns similar to BCR-ABL-positive ALL. Our aim was to evaluate new DNA-based measurable residual disease (MRD) tests detecting these fusions and IKZF1-deletions in comparison with conventional immunoglobulin/T-cell receptor (Ig/TCR) markers. Methods Precise genomic breakpoints were defined from targeted or whole genome next generation sequencing for ABL-fusions and BCR-ABL1. Quantitative PCR assays were designed and used to re-measure MRD in remission bone marrow samples previously tested using Ig/TCR markers. All MRD testing complied with EuroMRD guidelines. Results ABL-class patients had 46% 5year event-free survival and 79% 5year overall survival. All had sensitive fusion tests giving high concordance between Ig/TCR and ABL-class fusion results (21 patients, n = 257 samples, r2 = 0.9786, P IKZF1-deletion results (9 patients, n = 143 samples, r2 = 0.9661, P BCR-ABL1 patients, Ig/TCR and BCR-ABL1 tests were discordant in 32% (40 patients, n = 346 samples, r2 = 0.4703, P IKZF1-deletion results were closer to Ig/TCR (25 patients, n = 176, r2 = 0.8631, P Conclusions MRD monitoring based on patient-specific assays detecting gene fusions or recurrent assays for IKZF1-deletions is feasible and provides good alternatives to Ig/TCR tests to monitor MRD in ABL-class ALL.

Background While current chemotherapy has increased cure rates for children with acute lymphoblastic leukaemia (ALL), the largest number of relapsing patients are still stratified as medium risk (MR) at diagnosis (50–60%). This highlights an opportunity to develop improved relapse-prediction models for MR patients. We hypothesised that bone marrow from MR patients who eventually relapsed would regrow faster in a patient-derived xenograft (PDX) model after induction chemotherapy than samples from patients in long-term remission. Methods Diagnostic bone marrow aspirates from 30 paediatric MR-ALL patients (19 who relapsed, 11 who experienced remission) were inoculated into immune-deficient (NSG) mice and subsequently treated with either control or an induction-type regimen of vincristine, dexamethasone, and L-asparaginase (VXL). Engraftment was monitored by enumeration of the proportion of human CD45+ cells (%huCD45+) in the murine peripheral blood, and events were defined a priori as the time to reach 1% huCD45+, 25% huCD45+ (TT25%) or clinical manifestations of leukaemia (TTL). Results The TT25% value significantly predicted MR patient relapse. Mutational profiles of PDXs matched their tumours of origin, with a clonal shift towards relapse observed in one set of VXL-treated PDXs. Conclusions In conclusion, establishing PDXs at diagnosis and subsequently applying chemotherapy has the potential to improve relapse prediction in paediatric MR-ALL.

SummaryAccelerating cures for children with cancer remains an immediate challenge due to 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) from high-risk childhood cancers, many refractory to current standard-of-care treatments. Here, we genomically characterize 261 PDX models from 29 unique pediatric cancer malignancies and demonstrate faithful recapitulation of histologies, subtypes, and refine our understanding of relapsed disease. Expression and mutational signatures are used to classify tumors forTP53andNF1inactivation, as well as impaired DNA repair. We anticipate that these data will serve as a resource for pediatric oncology drug development and guide rational clinical trial design for children with cancer.HighlightsMultiplatform genomic analysis defines landscape of 261 pediatric cancer patient derived xenograft (PDX) modelsPediatric patient derived xenografts faithfully recapitulate relapsed diseaseInferredTP53pathway inactivation correlates with pediatric cancer copy number burdenSomatic mutational signatures predict impaired DNA repair across multiple histologies

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., Graphical Abstract, In Brief Rokita et. al provide an extensively annotated genomic dataset of somatic oncogenic regulation across 37 distinct pediatric malignancies. The 261 patient-derived xenograft models are available to the scientific community, and the genomic annotations will enable rational preclinical agent prioritization and acceleration of therapeutic targets for early-phase pediatric oncology clinical trials.

Relapse in pediatric T-cell acute lymphoblastic leukemia (T-ALL) remains a significant clinical problem and is thought to be associated with clonal selection during treatment. In this study we used an established pre-clinical model of induction therapy to increase our understanding of the effect of engraftment and chemotherapy on clonal selection and acquisition of drug resistance in vivo. Immune-deficient mice were engrafted with patient diagnostic specimens and exposed to a repeated combination therapy consisting of vincristine, dexamethasone, L-asparaginase and daunorubicin. Any re-emergence of disease following therapy was shown to be associated with resistance to dexamethasone, no resistance was observed to the other three drugs. Immunoglobulin/T-cell receptor gene rearrangements closely matched those in respective diagnosis and relapse patient specimens, highlighting that these clonal markers do not fully reflect the biological changes associated with drug resistance. Gene expression profiling revealed the significant underlying heterogeneity of dexamethasone-resistant xenografts. Alterations were observed in a large number of biological pathways, yet no dominant signature was common to all lines. These findings indicate that the biological changes associated with T-ALL relapse and resistance are stochastic and highly individual, and underline the importance of using sophisticated molecular techniques or single cell analyses in developing personalized approaches to therapy.

The heterodimeric transcription elongation factor Spt4/Spt5 (Spt4/5) tightly associates with RNAPII to regulate both transcriptional elongation and co-transcriptional pre-mRNA processing; however, the mechanisms by which Spt4/5 acts are poorly understood. Recent studies of the human and Drosophila Spt4/5 complexes indicate that they can bind nucleic acids in vitro. We demonstrate here that yeast Spt4/5 can bind in a sequence-specific manner to single stranded RNA containing AAN repeats. Furthermore, we show that the major protein determinants for RNA-binding are Spt4 together with the NGN domain of Spt5 and that the KOW domains are not required for RNA recognition. These findings attribute a new function to a domain of Spt4/5 that associates directly with RNAPII, making significant steps towards elucidating the mechanism behind transcriptional control by Spt4/5.

The effector protein AvrP is secreted by the flax rust fungal pathogen (Melampsora lini) and recognized specifically by the flax (Linum usitatissimum) P disease resistance protein, leading to effector-triggered immunity. To investigate the biological function of this effector and the mechanisms of specific recognition by the P resistance protein, we determined the crystal structure of AvrP. The structure reveals an elongated zinc-finger-like structure with a novel interleaved zinc-binding topology. The residues responsible for zinc binding are conserved in AvrP effector variants and mutations of these motifs result in a loss of P-mediated recognition. The first zinc-coordinating region of the structure displays a positively charged surface and shows some limited similarities to nucleic acid-binding and chromatin-associated proteins. We show that the majority of the AvrP protein accumulates in the plant nucleus when transiently expressed in Nicotiana benthamiana cells, suggesting a nuclear pathogenic function. Polymorphic residues in AvrP and its allelic variants map to the protein surface and could be associated with differences in recognition specificity. Several point mutations of residues on the non-conserved surface patch result in a loss of recognition by P, suggesting that these residues are required for recognition.

Introduction: While children diagnosed with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, the outcome for certain high-risk pediatric ALL subtypes as well as adult ALL remains poor. Several standard-of-care drugs used in multi-agent treatment protocols for ALL (including vincristine, daunorubicin and methotrexate) are substrates for the ATP-dependent drug efflux pump P-glycoprotein (P-gp), encoded by the ABCB1 gene, although there are limited reports of ABCB1 gene expression being associated with poor outcome in ALL (Carrillo et al, Hematol. 22:286-91, 2017). A previously identified high-risk subtype of T-ALL (early T-cell precursor ALL, ETP-ALL) characterized by poor early response to conventional induction treatment, expresses significantly higher levels of the ABCB1 gene compared with typical T-ALL (1.97-fold; false discovery rate=0.0026; P=0.00029; Zhang et al, Nature 481:157-63, 2012). E7130 is a novel anti-microtubule drug that is a less potent substrate for P-gp compared with other anti-microtubule drugs such as vincristine, and it has shown significant preclinical activity against patient-derived xenograft (PDX) models of adult malignancies. Therefore, it was of interest for the Pediatric Preclinical Testing Consortium (PPTC) to test the in vivo activity of E7130 against its PDX models of pediatric ETP-ALL. Methods: ABCB1 mRNA expression in ALL PDXs was quantified by RNAseq (https://pedcbioportal.org) and qRT-PCR. P-gp protein expression was assessed by immunoblotting, while its activity was measured by the Rhodamine-123 efflux assay in the absence or presence of the P-gp inhibitor tariquidar. E7130 and vincristine were evaluated in vivo against 6 ETP-ALL PDXs. Each PDX was inoculated into 6-8 immune-deficient (NSG) mice per treatment group (2-5 x 106 cells/mouse). Engraftment and drug responses were evaluated by enumerating the proportion of human CD45+ cells in the peripheral blood (%huCD45+) at weekly intervals. E7130 was tested at 2 dose levels (0.09 and 0.135 mg/kg IV), while vincristine was evaluated at 1 mg/kg IP. Both drugs were administered weekly x 3. Events were defined as the %huCD45+ ≥25%. Drug efficacy was assessed by event-free survival of treated (T) and control (C) groups by T-C, T/C and stringent objective response criteria (Houghton et al, Pediatr Blood Cancer 49:928-40, 2007). Results: RNAseq analysis of the 6 ETP-ALL PDXs in the PPTC panel of 90 pediatric ALL PDXs revealed 3 with high ABCB1 expression (FPKM 7.1-13.6; ETP-2, -3 and -6) and 3 with low expression (FPKM 0-0.15; ETP-1, -4 and -5), which was confirmed by qRT-PCR and immunoblotting. Moreover, high levels of tariquidar-sensitive Rhodamine-123 efflux activity were confirmed in the 2 high ABCB1 expressing PDXs tested (ETP-2 and -3). E7130 was generally well tolerated in NSG mice, with maximum average weight loss of 2.7-17.6% in the groups treated with the highest dose compared with 3.2-12.7% in the vincristine treated groups. E7130 (0.09 mg/kg) significantly (P Conclusions: E7130 exhibits significant in vivo activity against pediatric ETP-ALL PDXs, regardless of their levels of ABCB1 expression. Our results support further evaluation of E7130 in pediatric ALL to determine whether it represents an alternative treatment option in ALL with high ABCB1 expression. (Supported by NCI Grants CA199000 and CA199922) Disclosures No relevant conflicts of interest to declare.

alpha-Hemoglobin (alphaHb) stabilizing protein (AHSP) is expressed in erythropoietic tissues as an accessory factor in hemoglobin synthesis. AHSP forms a specific complex with alphaHb and suppresses the heme-catalyzed evolution of reactive oxygen species by converting alphaHb 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 alphaHb 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 alphaHb. In complex with alphaHb, AHSP Pro(30) adopts a cis-peptidyl conformation and makes contact with the N terminus of helix G in alphaHb. Mutations that stabilize the cis-peptidyl conformation of free AHSP, also enhance the alphaHb conversion activity. These findings suggest that AHSP loop 1 can transmit structural changes to the heme pocket of alphaHb, and, more generally, highlight the importance of cis-peptidyl prolyl residues in defining the conformation of regulatory protein loops.

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 sequence-specificity of four VapC proteins from two different organisms (VapCPAE0151 and VapCPAE2754 from Pyrobaculum aerophilum, and VapCRv0065 and VapCRv0617 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., Nucleic Acids Research, 40 (14), ISSN:1362-4962, ISSN:0301-5610