Fitting Correlated Hadron Mass Spectrum Data

We discuss fitting hadronic Green functions versus time $t$ to extract mass values in quenched lattice QCD. These data are themselves strongly correlated in $t$. With only a limited number of data samples, the method of minimising correlated $\chi^2$ is unreliable. We explore several methods of modelling the correlations among the data set by a few parameters which then give a stable and sensible fit even if the data sample is small. In particular these models give a reliable estimate of the goodness of fit.Comment: 14 pages, Latex text, followed by 3 postscript figures in self-unpacking file. Also available at ftp://suna.amtp.liv.ac.uk/pub/cmi/corfit

Quenched Hadrons using Wilson and O(a)-Improved Fermion Actions at beta=6.2

We present the first study of the light hadron spectrum and decay constants for quenched QCD using an O(a)-improved nearest-neighbour Wilson fermion action at \beta=6.2. We compare the results with those obtained using the standard Wilson fermion action, on the same set of 18 gauge field configurations of a 24^3 times 48 lattice. For pseudoscalar meson masses in the range 330-800 MeV, we find no significant difference between the results for the two actions. The scales obtained from the string tension and mesonic sector are consistent, but differ from that derived from baryon masses. The ratio of the pseudoscalar decay constant to the vector meson mass is roughly independent of quark mass as observed experimentally, and in approximate agreement with the measured value.Comment: 11 page

A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify additional therapeutic targets in AML, we optimize a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. We identify 492 AML-specific cell-essential genes, including several established therapeutic targets such as $\textit{DOT1L}$, $\textit{BCL2}$, and $\textit{MEN1}$, and many other genes including clinically actionable candidates. We validate selected genes using genetic and pharmacological inhibition, and chose $\textit{KAT2A}$ as a candidate for downstream study. $\textit{KAT2A}$ inhibition demonstrated anti-AML activity by inducing myeloid differentiation and apoptosis, and suppressed the growth of primary human AMLs of diverse genotypes while sparing normal hemopoietic stem-progenitor cells. Our results propose that KAT2A inhibition should be investigated as a therapeutic strategy in AML and provide a large number of genetic vulnerabilities of this leukemia that can be pursued in downstream studies.This work was funded by the Kay Kendall Leukaemia Fund (KKLF) and the Wellcome Trust (WT098051). G.S.V. is funded by a Wellcome Trust Senior Fellowship in Clinical Science (WT095663MA) and work in his laboratory is funded by Bloodwise. C.P. is funded by a Kay Kendall Leukaemia Fund Intermediate Fellowship (KKL888)

ASXL2 is essential for haematopoiesis and acts as a haploinsufficient tumour suppressor in leukemia

Additional sex combs-like (ASXL) proteins are mammalian homologues of additional sex combs (Asx), a regulator of trithorax and polycomb function in Drosophila. While there has been great interest in ASXL1 due to its frequent mutation in leukemia, little is known about its paralog ASXL2, which is frequently mutated in acute myeloid leukemia patients bearing the RUNX1-RUNX1T1 (AML1-ETO) fusion. Here we report that ASXL2 is required for normal haematopoiesis with distinct, non-overlapping effects from ASXL1 and acts as a haploinsufficient tumour suppressor. While Asxl2 was required for normal haematopoietic stem cell self-renewal, Asxl2 loss promoted AML1-ETO leukemogenesis. Moreover, ASXL2 target genes strongly overlapped with those of RUNX1 and AML1-ETO and ASXL2 loss was associated with increased chromatin accessibility at putative enhancers of key leukemogenic loci. These data reveal that Asxl2 is a critical regulator of haematopoiesis and mediates transcriptional effects that promote leukemogenesis driven by AML1-ETO

Hemopoietic-specific Sf3b1-K700E knock-in mice display the splicing defect seen in human MDS but develop anemia without ring sideroblasts.

Heterozygous somatic mutations affecting the spliceosome gene SF3B1 drive age-related clonal hematopoiesis, myelodysplastic syndromes (MDS) and other neoplasms. To study their role in such disorders, we generated knock-in mice with hematopoietic-specific expression of Sf3b1-K700E, the commonest type of SF3B1 mutation in MDS. Sf3b1K700E/+ animals had impaired erythropoiesis and progressive anemia without ringed sideroblasts, as well as reduced hematopoietic stem cell numbers and host-repopulating fitness. To understand the molecular basis of these observations, we analyzed global RNA splicing in Sf3b1K700E/+ hematopoietic cells. Aberrant splicing was associated with the usage of cryptic 3' splice and branchpoint sites, as described for human SF3B1 mutants. However, we found a little overlap between aberrantly spliced mRNAs in mouse versus human, suggesting that anemia may be a consequence of globally disrupted splicing. Furthermore, the murine orthologues of genes associated with ring sideroblasts in human MDS, including Abcb7 and Tmem14c, were not aberrantly spliced in Sf3b1K700E/+ mice. Our findings demonstrate that, despite significant differences in affected transcripts, there is overlap in the phenotypes associated with SF3B1-K700E between human and mouse. Future studies should focus on understanding the basis of these similarities and differences as a means of deciphering the consequences of spliceosome gene mutations in MDS

R.I.S.C.L: A Holistic Molecular Diagnostic Tool for Myeloid Malignancies

The genomic landscapes of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myeloproliferative disorders (MPD) and other related myeloid malignancies are now amongst the best characterized cancer genomes. These malignancies share most of their somatic driver mutations, many of which have therapeutic and prognostic significance (Patel et al, NEJM 2012). Patient prognostication and clinical decision-making can be greatly facilitated by testing for these mutations in parallel with established diagnostic assays. Here, we describe and validate RISCL (Rearrangements, Indels, Substitutions, Copy number and Loss-of-heterozygosity), a novel methodological and bioinformatic tool for the molecular diagnosis of myeloid malignancies. This tool employs targeted DNA capture to simultaneously: 1) identify coding mutations in 49 genes, 2) detect the four most important translocations in AML and 3) derive genome-wide copy number and zygosity data. Samples & methods 1. Samples Genomic DNA was extracted from bone marrow samples of 62 patients with AML (n=86 samples, including 24 remission samples) and 68 patients with MDS; and from blood granulocytes and mononuclear cells from 5 cord blood samples and 18 adults with normal hematopoiesis. 2. cRNA baits and sequencing The bait library (Agilent) contained 53,613 probes to capture: 1) all exons from 49 genes 2) intronic breakpoint sites for PML-RARA, CBFb-MYH11 and RUNX1-RUNX1T1 and MLL breakpoints 3) 9958 SNPs (minor allele frequency 0.40-0.45) for genome wide copy number and zygosity analysis. Barcoded sequencing was performed using Illumina HiSeq 2000 (100bp paired-end). 3. Bioinformatic analysis We used bespoke bioinformatics for detecting coding substitutions and indels (MIDAS; Conte et al, Leukemia 2013), chromosomal translocations (SMALT-FIT), copy number analysis (Avadis software) and detection of specific mutations such as MLL-PTD and FLT3-ITD (in-house scripts). 4. Verification of results To validate the sensitivity and specificity of our approach, we compared our findings to conventional diagnostic data and are also validating 30% of randomly selected variants. Results A mean of 94% of targeted bases were covered at least by 30x. In AML samples, the four most common coding mutations identified affected NPM1 (n=10), CEBPA (n=8), IDH1 (n=8) and NRAS (n=7). By comparison to conventional diagnostics, we detected 5/5 IDH1R132, 4/4 CEBPA, 1/1 IDH2R172K and 8/9 NPM1 mutations. In MDS samples, the top four mutations affected TP53 (n=15), TET2 (n=13), SRSF2 (n=9), ASXL1 (n=8) and mutations affecting spliceosome genes (n=18) that were mutually exclusive, as previously described (Yoshida et al, Nature 2011). RISCL detected 100% of known translocations (28/28) in AML patients, namely CBFb-MYH11 (n=8/8), PML-RARA (n=9/9), RUNX1-RUNX1T1 (n=4/4) and rearrangements of MLL (n=7/7). In every case of MLL rearrangement the gene partner was identified and in one case with t(X;11) we identified a novel gene partner to MLL, DIAPH2. Furthermore, we identified one patient with an MLL rearrangement not identified at diagnosis. Copy number analysis efficiently detected known large chromosomal deletions or monosomies in chromosome 5 (18/18) and 7 (10/12). Overall 47/54 large deletions were detected using Avadis software. Furthermore in one MDS patient we were able to detect a submicroscopic heterozygous deletion in chromosome 4 which included TET2. However this method was much less sensitive for detecting trisomies (13/27 trisomies detected overall). The reasons for this disparity between detection of deletions and amplifications using a standardized depth of coverage algorithm are unclear, but may include subclonal mutations, selection of karyotypically abnormal cells during metaphase preparation or limitations of our bioinformatic analysis, which we are currently investigating. Figure 1 shows an example of the results of our holistic analysis using RISCL from an informative case of AML. In summary we describe RISCL, a novel powerful holistic NGS tool for detailed characterization of myeloid malignancies that can be used for patient stratification and a personalized approach to malignancy in the molecular era. The same approach can be extended to other malignancies