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Supplementary Figure 5 - PDF file 30K, Figure showing RAS expression in hematopoietic cancer cell lines and human pediatric AML samples

Supplementary Figure 4 - PDF file 64K, Data showing the molecular Analysis of AMLs from NrasG12D/+ Mice

Supplementary Figure 2 - PDF file 177K, Data showing survival and Hematologic Parameters of Nras heterozygous, hemizygous and homozygous mice

Supplementary Figure 1 - PDF file 150K, Figure showing the generation and Functional Validation of a Conditional Mutant Nrasflox Allele

Automated detection of the region of interest (ROI) is a critical step in the two-step classification system in several medical image applications. However, key information such as model parameter selection, image annotation rules, and ROI confidence score are essential but usually not reported. In this study, we proposed a practical framework of ROI detection by analyzing hip joints seen on 7399 anteroposterior pelvic radiographs (PXR) from three diverse sources. We presented a deep learning-based ROI detection framework utilizing a single-shot multi-box detector with a customized head structure based on the characteristics of the obtained datasets. Our method achieved average intersection over union (IoU) = 0.8115, average confidence = 0.9812, and average precision with threshold IoU = 0.5 (AP50) = 0.9901 in the independent testing set, suggesting that the detected hip regions appropriately covered the main features of the hip joints. The proposed approach featured flexible loose-fitting labeling, customized model design, and heterogeneous data testing. We demonstrated the feasibility of training a robust hip region detector for PXRs. This practical framework has a promising potential for a wide range of medical image applications.

Colorectal cancer (CRC) remains a major contributor to cancer-related mortality. LIN28A and LIN28B are highly related RNA-binding protein paralogs that regulate biogenesis of let-7 microRNAs and influence development, metabolism, tissue regeneration, and oncogenesis. Here we demonstrate that overexpression of either LIN28 paralog cooperates with the Wnt pathway to promote invasive intestinal adenocarcinoma in murine models. When LIN28 alone is induced genetically, half of the resulting tumors harbor Ctnnb1 (β-catenin) mutation. When overexpressed in ApcMin/+ mice, LIN28 accelerates tumor formation and enhances proliferation and invasiveness. In conditional genetic models, enforced expression of a LIN28-resistant form of the let-7 microRNA reduces LIN28-induced tumor burden, while silencing of LIN28 expression reduces tumor volume and increases tumor differentiation, indicating that LIN28 contributes to tumor maintenance. We detected aberrant expression of LIN28A and/or LIN28B in 38% of a large series of human CRC samples (n = 595), where LIN28 expression levels were associated with invasive tumor growth. Our late-stage CRC murine models and analysis of primary human tumors demonstrate prominent roles for both LIN28 paralogs in promoting CRC growth and progression and implicate the LIN28/let-7 pathway as a therapeutic target.

E-cadherin is a major homophilic cell-cell adhesion molecule that inhibits motility of individual cells on matrix. However, its contribution to migration of cells through cell-rich tissues is less clear. We developed an in vivo sensor of mechanical tension across E-cadherin molecules, which we combined with cell-type-specific RNAi, photoactivatable Rac, and morphodynamic profiling, to interrogate how E-cadherin contributes to collective migration of cells between other cells. Using the Drosophila ovary as a model, we found that adhesion between border cells and their substrate, the nurse cells, functions in a positive feedback loop with Rac and actin assembly to stabilize forward-directed protrusion and directionally persistent movement. Adhesion between individual border cells communicates direction from the lead cell to the followers. Adhesion between motile cells and polar cells holds the cluster together and polarizes each individual cell. Thus, E-cadherin is an integral component of the guidance mechanisms that orchestrate collective chemotaxis in vivo. © 2014 Elsevier Inc.

Two interleukin-7 receptor mutants identified in human early T cell precursor leukemia are sufficient to induce disease in mice when expressed in primitive, Arf-null thymocytes., Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) exhibits lymphoid, myeloid, and stem cell features and is associated with a poor prognosis. Whole genome sequencing of human ETP-ALL cases has identified recurrent mutations in signaling, histone modification, and hematopoietic development genes but it remains to be determined which of these abnormalities are sufficient to initiate leukemia. We show that activating mutations in the interleukin-7 receptor identified in human pediatric ETP-ALL cases are sufficient to generate ETP-ALL in mice transplanted with primitive transduced thymocytes from p19Arf−/− mice. The cellular mechanism by which these mutant receptors induce ETP-ALL is the block of thymocyte differentiation at the double negative 2 stage at which myeloid lineage and T lymphocyte developmental potential coexist. Analyses of samples from pediatric ETP-ALL cases and our murine ETP-ALL model show uniformly high levels of LMO2 expression, very low to undetectable levels of BCL11B expression, and a relative lack of activating NOTCH1 mutations. We report that pharmacological blockade of Jak–Stat signaling with ruxolitinib has significant antileukemic activity in this ETP-ALL model. This new murine model recapitulates several important cellular and molecular features of ETP-ALL and should be useful to further define novel therapeutic approaches for this aggressive leukemia.

The control of mRNA stability plays a central role in orchestrating gene-regulatory networks in hematopoietic cell growth, differentiation and tumorigenesis. HNRNPA0, which encodes an RNA-binding protein shown to regulate transcript stability via binding to the AU-rich elements of mRNAs, is located within the commonly deleted segment of 5q31.2 in myeloid neoplasms with a del(5q), and is expressed at haploinsufficient levels in these patients. We show that HNRNPA0 is normally highly expressed in hematopoietic stem cells and exhibits dynamic changes in expression during the course of differentiation. To model HNRNPA0 haploinsufficiency, we used RNAi interference in primary murine cells and an experimental cell system, and found that reduced Hnrnpa0 expression leads to a shift from monocytic towards granulocytic differentiation. Microarray-based global expression profiling revealed that Hnrnpa0 knockdown disproportionally impacts AU-rich containing transcripts and alters expression of myeloid specification genes. In therapy-related myeloid neoplasms with a del(5q), AU-rich containing mRNAs are enriched in transcripts that encode proteins associated with increased growth and proliferation. Our findings implicate haploinsufficiency of HNRNPA0 as one of the key initiating mutations in the pathogenesis of myeloid neoplasms with a del(5q), and suggest that therapies that target AU-rich elements warrant consideration in efforts to develop new mechanism-based treatment strategies.

Background: Breast cancer remains a considerable health concern, despite major advances in the treatment and prevention of breast cancer. Inhibition of estrogen receptor signaling is one of the most effective therapies for patients with hormone receptor positive breast cancer. However, even after chemotherapy and hormonal therapy, local recurrence occurs frequently, and undesirable side effects lead to drug discontinuation in an estimated 40% of patients on adjuvant therapy. Autologous fat transfer is an emerging therapy used in breast reconstruction to restore surgical deformities after lumpectomy, or to rebuild the breast following mastectomy. This is an attractive option for both radiated and non-radiated patients, as complication rates in both groups are exceedingly low. Fat is injected into the breast or surgical cavity to maximize its contact with local vascularized tissue, to promote re-vascularization and survival of the adipose tissue. Thus, fat cells are injected throughout the tissue surrounding the surgical margin. We hypothesize that harvested adipose tissue can be loaded with a lipophilic drug prior to its re-injection into the breast cavity and its surrounding tissue. With the release of drug from the transplanted adipose tissue over time, the local concentration of an anti-estrogen within the breast parenchyma would be increased and systemic toxicity may be minimized. Method: Human adipose tissue was acquired from patients undergoing liposuction for autologous grafting. Tissue was washed with warm PBS, divided into 0.5 mL aliquots, and incubated in complete DMEM (10% FBS) containing increasing concentrations of 4-OH tamoxifen (0, 10, 20, or 40 uM) or fulvestrant (0, 0.1, 0.5, or 1.0 uM) for 12 hours. Following incubation, adipose tissue was washed thoroughly and co-cultured with MCF7 cells for 96 hours. Adipocyte co-cultured MCF7 were then collected and evaluated for proliferation, viability and effects on estrogen receptor signaling and compared to MCF7 directly exposed to anti-estrogens. Results: When co-culturing tamoxifen- or fulvestrant-loaded human adipocytes with MCF7 breast cancer cells in vitro, we observed a dose-dependent reduction in cell proliferation (tamoxifen, 60% reduction; fulvestrant, 40% reduction) and viability (20%), comparable to directly drugged media. Examination of the molecular response to fulvestrant demonstrated a dose dependent down regulation of ER protein expression and activity (e.g. PgR and Cyclin D1). Currently, we are characterizing the pharmacokinetics of anti-estrogen uptake and release in both in vitro and in vivo. Summary: Our preliminary data suggest that co-culturing human adipocytes with an anti-estrogen results in tumor growth inhibition and abrogation of estrogen receptor signaling. Human adipocytes used to fill the tumor bed cavity or mastectomy pocket could therefore be used as a vehicle to deliver anti-estrogens to residual tumor cells and prevent tumor regrowth. This method may be a novel technology to combine reconstructive surgery and anti-cancer therapy and prevention. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-16-01.

Breast cancer still continues to be a major cause of cancer related deaths in women, second only to lung cancer. Administering anti-estrogens and aromatase inhibitors, both in the adjuvant and metastatic settings, in estrogen receptor (ER) positive disease, is one of the most effective treatment strategies. However, prolonged exposure to these drugs leads to the emergence of resistance in about 40% of those who initially respond. Hence, the emphasis of a multitude of studies is to understand the underlying mechanisms of this acquired resistance and implementing means to overcome it. Recent studies have implicated epigenetic modulation of gene expression in the development of resistance to the anti-estrogen tamoxifen. A clinical trial conducted by our group has demonstrated the efficacy of combining the HDAC inhibitor vorinostat with tamoxifen in patients who had progressed on prior anti-estrogen therapies and showed a total 40% clinical benefit (19% objective response and 21% stable disease for more than 6 months). In an effort to identify patients most likely to benefit from this novel therapy, we sought to elucidate the mechanism behind the clinical efficacy of this combination. To this end, we have generated an in vitro tamoxifen resistant cell line (TAMR) by long-term exposure of MCF7 cells to 4-hydroxy tamoxifen. Significantly reduced anti-proliferative effect of tamoxifen and other anti-estrogens in TAMR compared to the sensitive MCF7 cells demonstrates the establishment of resistance to anti-estrogens in this cell line. In TAMR cells, addition of an HDAC inhibitor reverts resistance to tamoxifen. Although estrogen receptor (ER) expression in TAMR cells appears unaltered, the classical genomic signaling of ER in these resistant cells is suppressed and unresponsive to ligands, as deduced from transcription of ER response genes and luciferase assay of an ERE-luciferase construct. However, the ER remains important, since siRNA mediated depletion of ER inhibits cell growth in TAMR cells. Treatment with the dual EGFR/Her2 kinase inhibitor or an AKT inhibitor significantly inhibits the growth of these cells only when combined with tamoxifen, indicating the importance of crosstalk between pathways. Furthermore, Akt and mTOR protein and activities are down regulated by HDAC inhibition, which are further reduced when combined with tamoxifen. SiRNA mediated depletion of ER leads to reduced Akt and mTOR activities which suggests that ER may act as a driver, possibly through its non-genomic function at the plasma membrane activating members of the growth factor signaling pathways. Studies are ongoing to further characterize the interaction of HDAC and ER inhibition on these signaling pathways and to determine their significance to resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-13.

'Pre-leukaemic' mutations are thought to promote clonal expansion of haematopoietic stem cells (HSCs) by increasing self-renewal and competitiveness; however, mutations that increase HSC proliferation tend to reduce competitiveness and self-renewal potential, raising the question of how a mutant HSC can sustainably outcompete wild-type HSCs. Activating mutations in NRAS are prevalent in human myeloproliferative neoplasms and leukaemia. Here we show that a single allele of oncogenic Nras(G12D) increases HSC proliferation but also increases reconstituting and self-renewal potential upon serial transplantation in irradiated mice, all prior to leukaemia initiation. Nras(G12D) also confers long-term self-renewal potential to multipotent progenitors. To explore the mechanism by which Nras(G12D) promotes HSC proliferation and self-renewal, we assessed cell-cycle kinetics using H2B-GFP label retention and 5-bromodeoxyuridine (BrdU) incorporation. Nras(G12D) had a bimodal effect on HSCs, increasing the frequency with which some HSCs divide and reducing the frequency with which others divide. This mirrored bimodal effects on reconstituting potential, as rarely dividing Nras(G12D) HSCs outcompeted wild-type HSCs, whereas frequently dividing Nras(G12D) HSCs did not. Nras(G12D) caused these effects by promoting STAT5 signalling, inducing different transcriptional responses in different subsets of HSCs. One signal can therefore increase HSC proliferation, competitiveness and self-renewal through bimodal effects on HSC gene expression, cycling and reconstituting potential.

Biochemical properties of Ras oncoproteins and their transforming ability strongly support a dominant mechanism of action in tumorigenesis. However, genetic studies unexpectedly suggested that wild-type (WT) Ras exerts tumor suppressor activity. Expressing oncogenic Nras[superscript G12D] in the hematopoietic compartment of mice induces an aggressive myeloproliferative neoplasm that is exacerbated in homozygous mutant animals. Here, we show that increased Nras[superscript G12D] gene dosage, but not inactivation of WT Nras, underlies the aggressive in vivo behavior of Nras[superscript G12D over G12D] hematopoietic cells. Modulating Nras[superscript G12D] dosage had discrete effects on myeloid progenitor growth, signal transduction, and sensitivity to MAP-ERK kinase (MEK) inhibition. Furthermore, enforced WT N-Ras expression neither suppressed the growth of Nras-mutant cells nor inhibited myeloid transformation by exogenous Nras[superscript G12D]. Importantly, NRAS expression increased in human cancer cell lines with NRAS mutations. These data have therapeutic implications and support reconsidering the proposed tumor suppressor activity of WT Ras in other cancers., Pfizer Inc. (PD0325901), National Institutes of Health (U.S.) (Grant R37CA72614), National Institutes of Health (U.S.) (Grant P01CA40046), National Institutes of Health (U.S.) (Grant K08CA134649), Leukemia & Lymphoma Society of America (Specialized Center of Research Award LLS 7019-04)), American Lebanese Syrian Associated Charities

OBJECTIVE In mice, a lack of cryptochrome results in up-regulation of aldosterone production due to high expression of the 3β-hydroxysteroid dehydrogenases (HSD3β) gene. The HSD3β pathway might play a pivotal role in aldosterone synthesis. This study aimed to determine the association of HSD3β and HSD3β2 gene variations with primary aldosteronism in a Taiwanese population. METHOD In this case-control cohort, 688 consecutive ethnically matched unrelated individuals including 362 primary aldosteronism and 326 essential hypertension cases were recruited. Nineteen tag single-nucleotide polymorphisms (SNPs) across HSD3β1, HSD3β2, and CYP11β2 were genotyped. Expression of HSD3β mRNA and immunohistochemical stain of HSD3β in the specimens of aldosterone-producing adenoma (APA) was compared with that in nonfunctional incidentaloma. RESULTS The SNPs of rs12410453 A allele in HSD3β2 gene [odds ratio (OR) 1.92, 95% confidence interval (CI) 1.13-3.32, P=0.018] and rs6203 C allele in the HSD3β1 gene (OR 2.21, 95% CI 1.28-3.95, P=0.006) showed significant association with primary aldosteronism, with corresponding population attributable risk of 6.7 and 30.7%, respectively. Primary aldosteronism patients of non-CC in rs6203 and non-GA in rs12401453 had lower plasma aldosterone-to-renin ratio. A haplotype in a linkage disequilibrium block containing rs6203 associated significantly with serum potassium level (OR 1.24, 95% CI 1.02-1.24, P=0.026). The expressions of HSD3β1 mRNA, HSD3β2 mRNA and HSD3β protein were increased in APA, as compared to incidentaloma. CONCLUSION Risk-conferring genetic variations in the HSD3β gene influenced susceptibility of primary aldosteronism. Concomitant presence of rs6203 CC and rs12410453 GA genotypes synergistically increased aldosterone-to-renin ratio.

Reversing the aberrant biochemical output of oncogenic Ras proteins is one of the great challenges in cancer therapeutics; however, it is uncertain which Ras effectors are required for tumor initiation and maintenance. To address this question, we expressed oncogenic K-Ras(D12) proteins with "second site" amino acid substitutions that impair PI3 kinase/Akt or Raf/MEK/ERK activation in bone marrow cells and transplanted them into recipient mice. In spite of attenuated signaling properties, defective K-Ras oncoproteins initiated aggressive clonal T-lineage acute lymphoblastic leukemia (T-ALL). Murine T-ALLs expressing second site mutant proteins restored full oncogenic Ras activity through diverse mechanisms, which included acquiring novel somatic third site Kras(D12) mutations and silencing PTEN. T-ALL cell lines lacking PTEN had elevated levels of phosphorylated Akt, a gene expression pattern similar to human early T-cell precursor ALL, and were resistant to the potent and selective MEK inhibitor PD0325901. Our data, which demonstrate strong selective pressure to overcome the defective activation of PI3 kinase/Akt and Raf/MEK/ERK, implicate both Ras effector pathways as drivers of aberrant growth in T-ALL and further suggest that leukemia cells will deploy multiple mechanisms to develop resistance to targeted inhibitors in vivo.

The genetic basis of hypodiploid acute lymphoblastic leukemia (ALL), a subtype of ALL characterized by aneuploidy and poor outcome, is unknown. Genomic profiling of 124 hypodiploid ALL cases, including whole-genome and exome sequencing of 40 cases, identified two subtypes that differ in the severity of aneuploidy, transcriptional profiles and submicroscopic genetic alterations. Near-haploid ALL with 24-31 chromosomes harbor alterations targeting receptor tyrosine kinase signaling and Ras signaling (71%) and the lymphoid transcription factor gene IKZF3 (encoding AIOLOS; 13%). In contrast, low-hypodiploid ALL with 32-39 chromosomes are characterized by alterations in TP53 (91.2%) that are commonly present in nontumor cells, IKZF2 (encoding HELIOS; 53%) and RB1 (41%). Both near-haploid and low-hypodiploid leukemic cells show activation of Ras-signaling and phosphoinositide 3-kinase (PI3K)-signaling pathways and are sensitive to PI3K inhibitors, indicating that these drugs should be explored as a new therapeutic strategy for this aggressive form of leukemia.

Summary The TCF-1 and LEF-1 transcription factors are known to play critical roles in normal thymocyte development. Unexpectedly, we found that TCF-1-deficient ( Tcf7 −/− ) mice developed aggressive T cell malignancy, resembling human T cell acute lymphoblastic leukemia (T-ALL). LEF-1 was aberrantly upregulated in premalignant Tcf7 −/− early thymocytes and lymphoma cells. We further demonstrated that TCF-1 directly repressed LEF-1 expression in early thymocytes and that conditional inactivation of Lef1 greatly delayed or prevented T cell malignancy in Tcf7 −/− mice. In human T-ALLs, an early thymic progenitor (ETP) subtype was associated with diminished TCF7 expression, and two of the ETP-ALL cases harbored TCF7 gene deletions. We also showed that TCF-1 and LEF-1 were dispensable for T cell lineage commitment but instead were required for early thymocytes to mature beyond the CD4 − CD8 − stage. TCF-1 thus has dual roles, i.e., acting cooperatively with LEF-1 to promote thymocyte maturation while restraining LEF-1 expression to prevent malignant transformation of developing thymocytes.

To define the mutation spectrum in non-Down syndrome acute megkaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL leukemia samples. Our analysis identified a cryptic chromosome 16 inversion [inv(16)(p13.3q24.3)] in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling, and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

To the editor: Wnt signaling in hematopoietic cells and the bone marrow microenvironment plays a critical role in maintaining the pool of hematopoietic stem cells (HSCs) and in regulating differentiation.[1][1],[2][2] Wnt signaling is tightly regulated by the interplay of multiple cytoplasmic

Chromosome 7 deletions are highly prevalent in myelodysplastic syndrome (MDS) and likely contribute to aberrant growth through haploinsufficiency. We generated mice with a heterozygous germ line deletion of a 2-Mb interval of chromosome band 5A3 syntenic to a commonly deleted segment of human 7q22 and show that mutant hematopoietic cells exhibit cardinal features of MDS. Specifically, the long-term hematopoietic stem cell (HSC) compartment is expanded in 5A3+/del mice, and the distribution of myeloid progenitors is altered. 5A3+/del HSCs are defective for lymphoid repopulating potential and show a myeloid lineage output bias. These cell autonomous abnormalities are exacerbated by physiologic aging and upon serial transplantation. The 5A3 deletion partially rescues defective repopulation in Gata2 mutant mice. 5A3+/del hematopoietic cells exhibit decreased expression of oxidative phosphorylation genes, increased levels of reactive oxygen species, and perturbed oxygen consumption. These studies provide the first functional data linking 7q22 deletions to MDS pathogenesis. DOI: http://dx.doi.org/10.7554/eLife.07839.001, eLife digest Stem cells in the bone marrow are essential for creating new blood cells. Myelodysplastic syndrome (MDS) is a common type of blood cancer in the elderly that occurs when blood cells fail to develop normally. Depending on which types of blood cells are affected, individuals with MDS may bleed more easily, feel weak and tired, or be unable to effectively fight off infections. Animals and plants store their genetic information in the form of chromosomes. Humans have 23 pairs of chromosomes, with one copy inherited from the mother, and the other from the father. The bone marrow cells of many people with MDS delete a section from one of their copies of chromosome 7. As this section contains many different genes, it is difficult to fully understand which specific genes contribute to the development of MDS when one copy is lost. Wong et al. have now genetically engineered mice to lack a section of one of their copies of chromosome 7 that is often missing in patients with MDS. Bone marrow cells from these mice exhibit many of the same abnormalities found in human MDS. For example, most of the immature blood stem cells expand, but these stem cells do not correctly specialize into mature blood cells—in particular, not enough immune cells are produced. The developing blood cells also have problems expressing several genes, including one that helps to protect the cells from damaging molecules called reactive oxygen species. These problems worsen as the mice age. These mice provide the first evidence directly linking the missing section of chromosome 7 to abnormalities found in MDS patients. Future studies using the mice will advance our understanding of how the loss of this section of chromosome 7 interacts with other genes involved in MDS to alter the course of this disease and how it responds to treatment. DOI: http://dx.doi.org/10.7554/eLife.07839.002

An assessment of the equilibrium dynamics of biomolecular systems, and in particular their most cooperativefluctuations accessibleundernative state conditions, is a first step towards understanding molecular mechanisms relevant to biological function. We present a web-based system, oGNM that enables users to calculate online the shape and dispersion of normal modes of motion for proteins, oligonucleotides and their complexes, or associated biological units, using the Gaussian Network Model (GNM). Computations with the new engine are 5–6 orders of magnitude faster than those using conventional normal mode analyses. Two cases studies illustrate the utility of oGNM. The first shows that the thermal fluctuations predicted for 1250 non-homologous proteins correlate well with X-ray crystallographic data over a broad range [7.3–15 A u ] of inter-residue interaction cutoff distances and the correlations improve with increasing observation temperatures. The second study, focused on 64 oligonucleotides and oligonucleotide–protein complexes, shows that good agreement with experiments is achieved by representing each nucleotide by three GNM nodes (as opposed to one-node-per-residue in proteins) along with uniform interaction ranges for all components of the complexes. These results open the way to a rapid assessment of the dynamics of DNA/ RNA-containing complexes. The server can be accessed at http://ignm.ccbb.pitt.edu/GNM_Online_ Calculation.htm.

Motivation: Analysis of protein sequence and structure databases usually reveal frequent patterns (FP) associated with biological function. Data mining techniques generally consider the physicochemical and structural properties of amino acids and their microenvironment in the folded structures. Dynamics is not usually considered, although proteins are not static, and their function relates to conformational mobility in many cases. Results: This work describes a novel unsupervised learning approach to discover FPs in the protein families, based on biochemical, geometric and dynamic features. Without any prior knowledge of functional motifs, the method discovers the FPs for each type of amino acid and identifies the conserved residues in three protease subfamilies; chymotrypsin and subtilisin subfamilies of serine proteases and papain subfamily of cysteine proteases. The catalytic triad residues are distinguished by their strong spatial coupling (high interconnectivity) to other conserved residues. Although the spatial arrangements of the catalytic residues in the two subfamilies of serine proteases are similar, their FPs are found to be quite different. The present approach appears to be a promising tool for detecting functional patterns in rapidly growing structure databases and providing insights in to the relationship among protein structure, dynamics and function. Availability: Available upon request from the authors.

SummaryAlterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL.

PMCID: PMC4636050.-- et al., Spatial and temporal dissection of the genomic changes occurring during the evolution of human non-small cell lung cancer (NSCLC) may help elucidate the basis for its dismal prognosis.We sequenced 25 spatially distinct regions from seven operable NSCLCs and found evidence of branched evolution, with driver mutations arising before and after subclonal diversification. There was pronounced intratumor heterogeneity in copy number alterations, translocations, and mutations associated with APOBEC cytidine deaminase activity. Despite maintained carcinogen exposure, tumors from smokers showed a relative decrease in smoking-related mutations over time, accompanied by an increase in APOBEC-associated mutations. In tumors from former smokers, genome-doubling occurred within a smoking-signature context before subclonal diversification, which suggested that a long period of tumor latency had preceded clinical detection. The regionally separated driver mutations, coupled with the relentless and heterogeneous nature of the genome instability processes, are likely to confound treatment success in NSCLC., E.B. is a Rosetrees Trust fellow; M.J.H. has a Cancer Research UK fellowship; N.Mu. received funding from the Rosetrees Trust; M.G. is funded by the UK Medical Research Council; I.V. is funded by Spanish Ministerio de Economía y Competitividad subprograma Ramón y Cajal; R.C.R. and D.M.R. are partly funded by the Cambridge Biomedical Research Centre and Cancer Research UK Cancer Centre; P.V.L. is a postdoctoral researcher of the Research Foundation—Flanders (FWO); S.M.J. is a Wellcome Senior Fellow in Clinical Science; and C.S. is a senior Cancer Research UK clinical research fellow and is funded by Cancer Research UK, the Rosetrees Trust, European Union Framework Programme 7 (projects PREDICT and RESPONSIFY, ID:259303), the Prostate Cancer Foundation, the European Research Council and the Breast Cancer Research Foundation. This research is supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre.

Contains fulltext : 126772.pdf (Publisher’s version ) (Closed access) Somatic alterations of the lymphoid transcription factor gene PAX5 (also known as BSAP) are a hallmark of B cell precursor acute lymphoblastic leukemia (B-ALL), but inherited mutations of PAX5 have not previously been described. Here we report a new heterozygous germline variant, c.547G>A (p.Gly183Ser), affecting the octapeptide domain of PAX5 that was found to segregate with disease in two unrelated kindreds with autosomal dominant B-ALL. Leukemic cells from all affected individuals in both families exhibited 9p deletion, with loss of heterozygosity and retention of the mutant PAX5 allele at 9p13. Two additional sporadic ALL cases with 9p loss harbored somatic PAX5 substitutions affecting Gly183. Functional and gene expression analysis of the PAX5 mutation demonstrated that it had significantly reduced transcriptional activity. These data extend the role of PAX5 alterations in the pathogenesis of pre-B cell ALL and implicate PAX5 in a new syndrome of susceptibility to pre-B cell neoplasia.

Mutations that deregulate Notch1 and Ras/phosphoinositide 3 kinase (PI3K)/Akt signalling are prevalent in T-cell acute lymphoblastic leukaemia (T-ALL), and often coexist. Here we show that the PI3K inhibitor GDC-0941 is active against primary T-ALLs from wild-type and KrasG12D mice, and addition of the MEK inhibitor PD0325901 increases its efficacy. Mice invariably relapsed after treatment with drug-resistant clones, most of which unexpectedly had reduced levels of activated Notch1 protein, downregulated many Notch1 target genes, and exhibited cross-resistance to γ-secretase inhibitors. Multiple resistant primary T-ALLs that emerged in vivo did not contain somatic Notch1 mutations present in the parental leukaemia. Importantly, resistant clones upregulated PI3K signalling. Consistent with these data, inhibiting Notch1 activated the PI3K pathway, providing a likely mechanism for selection against oncogenic Notch1 signalling. These studies validate PI3K as a therapeutic target in T-ALL and raise the unexpected possibility that dual inhibition of PI3K and Notch1 signalling could promote drug resistance in T-ALL. Mutations that dysregulate Notch1 and Ras/PI3K signalling are common in T-cell acute lymphoblastic leukaemia; here, treatment with a PI3K inhibitor is shown to induce drug resistance that is associated with downregulation of activated Notch1 signalling, suggesting that inhibition of both Notch1 and PI3K could promote drug resistance. T-cell acute lymphoblastic leukaemia (T-ALL) is closely associated with mutations that deregulate Notch1 and Ras/phosphoinositide 3 kinase (PI3K) signalling. Kevin Shannon and colleagues show that treatment with GDC-0941 (pictilisib), a PI3K inhibitor that is advancing in clinical development, induces resistance associated with downregulation of activated Notch signalling, cross-resistance to γ-secretase inhibitors and upregulated PI3K signalling. Inhibition of Notch1 activates the PI3K pathway, suggesting that the rational strategy of concurrently targeting activated Notch1 and PI3K in T-ALL is likely to accelerate drug resistance.

Acute lymphoblastic leukemia (ALL) is the commonest childhood malignancy and is characterized by recurring structural genetic alterations. Previous studies of DNA methylation suggest epigenetic alterations may also be important, but an integrated genome-wide analysis of genetic and epigenetic alterations in ALL has not been performed. We analyzed 137 B-lineage and 30 T-lineage childhood ALL cases using microarray analysis of DNA copy number alterations and gene expression, and genome-wide cytosine methylation profiling using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. We found that the different genetic subtypes of ALL are characterized by distinct DNA methylation signatures that exhibit significant correlation with gene expression profiles. We also identified an epigenetic signature common to all cases, with correlation to gene expression in 65% of these genes, suggesting that a core set of epigenetically deregulated genes is central to the initiation or maintenance of lymphoid transformation. Finally, we identified aberrant methylation in multiple genes also targeted by recurring DNA copy number alterations in ALL, suggesting that these genes are inactivated far more frequently than suggested by structural genomic analyses alone. Together, these results demonstrate subtype- and disease-specific alterations in cytosine methylation in ALL that influence transcriptional activity, and are likely to exert a key role in leukemogenesis.

Pharmacogenomics (PGx) is the study of genetic variations in terms of their response to drugs. Variations in gene sequence or copy number may result in complete loss of function, partial decrease or increase in enzyme activity, or an altered affinity for substrates, which may in turn significantly impact a drug's efficacy. PGx studies are increasing in significance as precision medicine is becoming a reality in standard practice. Different technologies have been developed to measure the sequence variation and copy number variation (CNV) in the PGx genes. Among them, a complete sample-to-result PGx workflow solution using the QuantStudio™ 12k Flex Real-Time PCR System is the most notable high throughput solution and has broad adoption by advanced PGx laboratories. Both PGx SNP/INDEL genotyping assays on OpenArray™ plates and copy number analysis on 384-well plates can be performed on the QuantStudio™ 12k Flex System. Integrated analysis software translates genotyping and copy number assay results into star allele genotypes for ease of interpretation. Recently we have developed a next generation sequencing (NGS) based PGx research solution with increased flexibility on the assay targets and combined detection of SNP/INDEL genotyping and CNV using Ion AmpliSeq™ technology for low to medium throughput laboratories. With a highly multiplexed PGx research panel, we can profile a set of 136 genetic markers in 40 known PGx related genes and CYP2D6 copy number variation in a single reaction using Ion Torrent™ semiconductor sequencing. The number of genetic markers can be customized easily based on the user need. To systematically compare these two end-to-end PGx workflows, we collected buccal swab samples from 20 individuals and performed both QuantStudio™ based assays and PGM™ based Ion AmpliSeq™ PGx research assay on them. Both systems generated high quality results. Compared with OpenArray™ plate genotyping results and 384-plate CYP2D6 copy number assay results from the QuantStudio™ system, the Ion AmpliSeq™ PGx research solution demonstrated >99.9% genotyping concordance, 100% CYP2D6 gene CNV concordance, >99.7% reproducibility, Citation Format: Melvin S. Wei, Zhoutao Chen, Shann-Ching Chen, Manimozhi Manivannan, Emily Zeringer, Sunali Patel, Toinette Hartshorne, Guoying Liu, Fiona Hyland, Mark Andersen. A next-generation sequencing-based sample-to-result pharmacogenomics research solution enables both SNV and CNV detection at once. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2042.

Introduction: Despite risk stratification according to presenting clinical and genetic features, 10-25% of children with acute lymphoblastic leukemia (ALL) relapse, which is associated with a poor prognosis. Here, we sought to provide a comprehensive overview of the genetic alterations associated with relapse in ALL. Methods: We studied 93 children (27 female, 43 male) diagnosed with ALL (62 B-progenitor, 25 T-lineage) between 1987 and 2008 and treated on total therapy studies XI-XVI who experienced relapse and/or a second tumor. Age at diagnosis ranged from 3 months to 18 years. Median time to relapse was 3 years (range 3 months to 10 years). Seventy patients had a single relapse, 15 cases had 2 relapses, and 8 cases developed a second tumor of different lineage (B-cell lymphoma, chronic myeloid leukemia (n=1 each) and acute myeloid leukemia (n=6)). Diagnosis, relapse and matched normal samples (n=299) were studied using Affymetrix SNP 6.0 microarrays and whole genome or whole exome sequencing. Results: We found 2692 copy number aberrations (CNAs) with a median of 9 (range 0-109) in the diagnosis samples (n=91) compared to a median of 10 (range 0-112) in the relapse samples (n=89) and 12 (range 0-70) in subsequent samples (n=20). The number of CNAs did not differ significantly between diagnosis, relapse or subsequent samples. We identified a 7286 non-silent single nucleotide variants (SNVs) and small insertions or deletions (indels) in 5002 genes, 1392 of which were recurrent. The median number of variants was 12 (range 0-70) at diagnosis (n=91), 21 (range 0-858) at relapse (n=91; P=0.0029 v. diagnosis) and 60 (range 10-650) in subsequent samples (n=20; P The most frequently mutated genes were NOTCH1 (n=33), NRAS (n=24), CREBBP (n=20) and KRAS (n=16). Of the recurrently altered genes, only 87 genes were known to be affected in cancer (Cancer Gene Census, COSMIC database), of which 59 were affected in leukemia and lymphoma tissues, indicating that we have identified 1306 novel recurrently affected genes, most commonly C13orf40 and MKI67. Mutations in epigenetic regulators were particularly frequent, with genes mutated in at least 3 cases altered in over 60% of the cohort (e.g. CREBBP, EP300, MLL2, MLL3, KDM6A/B, CTCF, SETD2, TET2/3, and EZH2). Clonal evolution analyses showed multiple patterns of evolution, with relapses sharing either few or many variants with the diagnosis sample in a frequency that reflects both predominant clones and minor subclones propagating relapse. Variants in NOTCH1, NRAS, and CREBBP were preserved from a major clone at diagnosis in 4, 6, and 5 cases respectively, but acquired at relapse or grown out from a minor subclone at diagnosis in 3, 5, and 8 cases respectively. In contrast, variants in USH2A (n=4), FOXA1 (n=3), and purine/pyrimidine synthesis pathway genes NT5C2 (n=3), PRPS1 (n=3) and NT5C1B (n=1) were exclusively found in relapse samples. Notably, the NT5C2 mutations, which are thought to confer resistance to thiopurines, were subclonal at relapse in the majority of cases. We identified 13 cases (10 B-lineage, 3 T-lineage) in which the diagnosis and relapse were fully discordant for all CNAs and sequence mutations, only 4 of which showed a prolonged remission time (>5 years). This suggests that these patients developed a second primary malignancy and may be predisposed to leukemia development. Indeed, one case revealed focal amplifications on chromosome 1q21.1 encompassing the neuroblastoma breakpoint family genes, which are implicated in cancer development. Comprehensive germline analyses are underway. Conclusion: This study has provided detailed insight into the genetic basis of relapse, implicating multiple new genes and pathways involved in treatment resistance, demonstrating multiple patterns of clonal evolution, and revealing an unexpectedly high frequency of genetically discordant second malignancy in relapse in ALL. Disclosures Evans: Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Mullighan:Amgen: Honoraria, Speakers Bureau; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria; Loxo Oncology: Research Funding.

The BCL11B transcription factor is required for normal T-cell development, and has recently been implicated in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) induced by TLX overexpression or Atm deficiency. To comprehensively assess the contribution of BCL11B inactivation to human T-ALL, we performed DNA copy number and sequencing analyses of T-ALL diagnostic specimens, revealing monoallelic BCL11B deletions or missense mutations in 9% (n = 10 of 117) of cases. Structural homology modeling revealed that several of the BCL11B mutations disrupted the structure of zinc finger domains required for this transcription factor to bind DNA. BCL11B haploinsufficiency occurred across each of the major molecular subtypes of T-ALL, including early T-cell precursor, HOXA-positive, LEF1-inactivated, and TAL1-positive subtypes, which have differentiation arrest at diverse stages of thymocyte development. Our findings provide compelling evidence that BCL11B is a haploinsufficient tumor suppressor that collaborates with all major T-ALL oncogenic lesions in human thymocyte transformation.

Abstract 757 Acute Megakaryoblastic Leukemia (AMKL) accounts for ∼10% of childhood acute myeloid leukemia (AML). Although AMKL patients with down syndrome (DS-AMKL) have an excellent 5 year event-free survival (EFS), non-DS-AMKL patients have an extremely poor outcome with a 3 year EFS of less than 40%. With the exception of the t(1;22) translocation seen in infant non-DS-AMKL, little is known about the molecular genetic lesions that underlie this leukemia subtype. To define the landscape of mutations that occur in non-DS-AMKL, we performed transcriptome sequencing on diagnostic blasts from 14 cases (discovery cohort) using the illumina platform. Our results identified chromosomal rearrangements resulting in the expression of novel fusion transcripts in 12/14 cases. Remarkably, in 7/14 cases we detected an inversion on chromosome 16 [inv(16)(p13.3;q24.3)] that resulted in the juxtaposition of the CBFA2T3, a member of the ETO family of transcription factors, next to GLIS2 resulting in a CBFA2T3-GLIS2 chimeric gene encoding an in frame fusion protein. 6 cases in the discovery cohort fused exon 10 of CBFA2T3 to exon 3 of GLIS2, while 1 case carried a larger product that fused exon 11 of CBFA2T3 to exon 1 of GLIS2. Both products retain the 3 CBFA2T3 N-terminal nervy homology regions that mediate protein interactions, and the 5 GLIS2 C-terminal zinc finger domains that bind the Glis DNA consensus sequence, along with one of its N-terminal transcriptional regulatory domains. GLIS2 is a member of the GLI super family of transcription factors and has been demonstrated to play a role in regulating expression of GLI target genes as well as inhibiting WNT signaling through the binding of beta catenin. Although GLIS2 is not normally expressed in hematopoietic cells, the translocation results in high level expression of the CBFA2T3-GLIS2 fusion protein. In addition to CBFA2T3-GLIS2, chimeric transcripts were detected in 6/7 cases that lacked evidence of the inv(16)(p13.3;q24.3). Specifically, we detected GATA2-HOXA9, MN1-FLI1, NIPBL-HOXB9, NUP98-KDM5A, GRB10-SDK1 and C8orf76-HOXA11AS, each in an individual case. Importantly, several of the genes involved in these translocations either play a direct role in normal megakaryocytic differentiation (GATA2 and FLI1), or have been previously shown to be involved in leukemogenesis (HOXA9, MN1, HOXB9). Evaluation of a recurrency cohort of 42 samples including 14 additional pediatric cases and 28 adult cases by RT-PCR revealed 4 additional pediatric samples carrying CBFA2T3-GLIS2 for an overall frequency of 39% in pediatric AMKL. In addition to these somatic structural variations, we also identified mutations in genes previously shown to play a role in megakaryoblastic leukemia including activating mutations in JAK2 and MPL (36%). To gain insight into the mechanism whereby CBFA2T3-GLIS2 promotes leukemogenesis, we introduced the fusion into murine hematopoietic cells and assessed its effect on in vitro colony replating as a surrogate measure of self-renewal. Hematopoietic cells transduced with a mCherry expressing retroviral vector failed to form colonies after the second replating. By contrast, expression of either wild-type GLIS2 or the CBFA2T3-GLIS2 fusion resulted in a marked increase in the self-renewal capacity, with colony formation persisting through eight replatings. Immunophenotypic analysis of the CBFA2T3-GLIS2 expressing colonies revealed evidence of megakaryocytic differentiation. Importantly, the CBFA2T3-GLIS2 cells remained growth factor dependent suggesting that cooperating mutations in growth factor signaling pathways are required for full leukemic transformation. Taken together these data identify a novel cryptic inv(16)-encoded CBFA2T3-GLIS2 fusion protein as a recurrent driver mutation in approximately 40% of non-infant pediatric non-DS-AMKLs. Moreover, the majority of pediatric cases that lacked this lesion were shown by transcriptome sequence analysis to contain other chromosomal rearrangements that encoded fusion proteins that directly alter megakaryocytic differentiation and/or myeloid cell growth. The alteration of a key transcriptional regulator within the hedgehog signaling pathways in a substantial percentage of pediatric AMKL raises the possibility that inhibition of this pathway may have a therapeutic benefit in this aggressive form of AML. *TAG and ALG contributed equally to this work. Disclosures: Biondi: BMS, Novartis, Micromed: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ravandi:Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria. Kantarjian:Novartis: Consultancy, Research Funding; Pfizer: Research Funding; BMS: Research Funding. Doehner:Hoffmann La Roche: Honoraria.

Motivation: The yeast Saccharomyces cerevisiae is the first eukaryotic organism to have its genome completely sequenced. Since then, several large-scale analyses of the yeast genome have provided extensive functional annotations of individual genes and proteins. One fundamental property of a protein is its subcellular localization, which provides critical information about how this protein works in a cell. An important project therefore was the creation of the yeast GFP fusion localization database by the University of California, San Francisco, USA (UCSF). This database provides localization data for 75% of the proteins believed to be encoded by the yeast genome. These proteins were classified into 22 distinct subcellular location categories by visual examination. Based on our past success at building automated systems to classify subcellular location patterns in mammalian cells, we sought to create a similar system for yeast. Results: We developed computational methods to automatically analyze the images created by the UCSF yeast GFP fusion localization project. The system was trained to recognize the same location categories that were used in that study. We applied the system to 2640 images, and the system gave the same label as the previous assignments to 2139 images (81%). When only the highest confidence assignments were considered, 94.7% agreement was observed. Visual examination of the proteins for which the two approaches disagree suggests that at least some of the automated assignments may be more accurate. The automated method provides an objective, quantitative and repeatable assignment of protein locations that can be applied to new collections of yeast images (e.g. for different strains or the same strain under different conditions). It is also important to note that this performance could be achieved without requiring colocalization with any marker proteins. Availability: The original images analyzed in this article are available at http://yeastgfp.ucsf.edu, and source code and results are available at http://murphylab.web.cmu.edu/software Contact: murphy@cmu.edu

Despite extensive investigation into the molecular basis of tumor development, colorectal cancer (CRC) remains a major contributor to cancer-related mortality. LIN28A and LIN28B are highly related RNA-binding proteins that influence development, tissue regeneration and oncogenesis. Here we demonstrate that overexpression of LIN28 drives invasive intestinal adenocarcinoma and cooperates with the Wnt pathway to promote tumor initiation and progression in murine models. Importantly, conditional withdrawal of LIN28 reduced tumor volume and increased tumor differentiation, indicating that LIN28 contributes to tumor maintenance. Furthermore, co-expression of a LIN28-resistant form of the let-7 microRNA reduced tumor burden. We detected aberrant expression of LIN28 in 38% of a large series of tumor samples from human CRC, and LIN28 expression levels were associated with invasive tumor growth pattern. Our robust late-stage CRC murine model demonstrates strong roles for LIN28 in promoting CRC initiation, growth and progression, and suggests the therapeutic potential in targeting the LIN28/let-7 pathway. Citation Format: Ho-Chou Tu, Sarah Schwitalla, Zhirong Qian, Grace LaPier, Alena Yermalovich, Yuan-Chieh Ku, Shann-Ching Chen, Srinivas R. Viswanathan, Hao Zhu, Reiko Nishihara, Kentaro Inamura, Sun A. Kim, Reppi Morikawa, Kosuke Mima, Yasutaka sukawa, Juhong Yang, Gavin Meredith, Charles S. Fuchs, Shuji Ogino, George Q. Daley. LIN28 cooperates with Wnt signaling to drive invasive intestinal and colorectal adenocarcinoma in mouse and human. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2300. doi:10.1158/1538-7445.AM2015-2300

Background Knowledge of the subcellular location of a protein is critical to understanding how that protein works in a cell. This location is frequently determined by the interpretation of fluorescence microscope images. In recent years, automated systems have been developed for consistent and objective interpretation of such images so that the protein pattern in a single cell can be assigned to a known location category. While these systems perform with nearly perfect accuracy for single cell images of all major subcellular structures, their ability to distinguish subpatterns of an organelle (such as two Golgi proteins) is not perfect. Our goal in the work described here was to improve the ability of an automated system to decide which of two similar patterns is present in a field of cells by considering more than one cell at a time. Since cells displaying the same location pattern are often clustered together, considering multiple cells may be expected to improve discrimination between similar patterns. Results We describe how to take advantage of information on experimental conditions to construct a graphical representation for multiple cells in a field. Assuming that a field is composed of a small number of classes, the classification accuracy can be improved by allowing the computed probability of each pattern for each cell to be influenced by the probabilities of its neighboring cells in the model. We describe a novel way to allow this influence to occur, in which we adjust the prior probabilities of each class to reflect the patterns that are present. When this graphical model approach is used on synthetic multi-cell images in which the true class of each cell is known, we observe that the ability to distinguish similar classes is improved without suffering any degradation in ability to distinguish dissimilar classes. The computational complexity of the method is sufficiently low that improved assignments of classes can be obtained for fields of twelve cells in under 0.04 second on a 1600 megahertz processor. Conclusion We demonstrate that graphical models can be used to improve the accuracy of classification of subcellular patterns in multi-cell fluorescence microscope images. We also describe a novel algorithm for inferring classes from a graphical model. The performance and speed suggest that the method will be particularly valuable for analysis of images from high-throughput microscopy. We also anticipate that it will be useful for analyzing the mixtures of cell types typically present in images of tissues. Lastly, we anticipate that the method can be generalized to other problems.

A protein molecule is made of a long chain of amino acid sequences that fold into a complex three-dimensional structure. It is often the geometrical shapes that determine the protein functions. In molecular biology, researchers use sequence alignment and structure matching to compare the similarity among proteins. Considering proteins as 3D structures, we propose a novel algorithm to identify geometry-based features to retrieve similar proteins without having to deal with complex chemical characteristics and biological properties. A Web-based Dali server, which performs well in three-dimensional structure matching, is used as the ground truth to evaluate our algorithm. Our system performs close to the ground truth with much simplicity and efficiency.

BCR-ABL1-like, or “Ph-like” B-progenitor acute lymphoblastic leukemia (B-ALL) constitutes up to 15% of childhood and 30% of adult ALL, and is characterized by a gene expression profile similar to BCR-ABL1 ALL, alteration of IKZF1, and poor outcome. A pilot next-generation sequencing study identified kinase activating alterations in 15 Ph-like ALL cases. The goals of this study were to define the genomic landscape of Ph-like ALL in children and young adults, and to examine the utility of tyrosine kinase inhibitors (TKIs) in patients harboring genetic alterations activating kinase signaling. We studied 1665 B-ALL cases, including 309 childhood standard risk (10.8% Ph-like), 826 childhood high risk (14% Ph-like), 370 adolescent (16-21 years, 21% Ph-like) and 160 young adult (21-39 years; 26% Ph-like) cases. Approximately 50% of Ph-like cases harbored a CRLF2 rearrangement (IGH-CRLF2 or P2RY8-CRLF2). Next-generation sequencing was performed for 160 non-CRLF2 expressing Ph-like cases, including mRNA-seq (141 cases), whole genome sequencing (30 cases) and/or exome sequencing (12 cases). Fusion transcripts were identified using CICERO, a novel mRNA-seq assembly-based structural variation detection method. Over 100 chimeric in-frame fusions were identified, including 29 involving 12 tyrosine kinase or cytokine receptor genes, 15 of which were recurrent: JAK2 (10 partners), ABL1 (6), ABL2 (3), PDGFRB (3), CSF1R, TYK2, NTRK3, PTK2B, IL2RB (1 partner each), and rearrangements of EPOR into the IGH and IGK loci. Together, these rearrangements were present in ∼30% of Ph-like ALL cases. Additional sequence and structural alterations activating kinase signaling were identified in ∼10% of cases (e.g IL7R, FLT3, SH2B3). Despite the diversity of kinase alterations, the majority are predicted to respond to a limited number of TKIs, but many are novel or have not been tested in suitable preclinical models of ALL. We show that expression of RCSD1-ABL1, RANBP2-ABL1, ZMIZ1-ABL1, RCSD1-ABL2, SSBP2-CFS1R and PAX5-JAK2 in Ba/F3 and primary mouse pre-B cultures induces cytokine-independent proliferation and constitutive activation of JAK/STAT signaling. Furthermore, the ABL1, ABL2 and CSF1R fusions were sensitive to dasatinib (IC50 range 1-2nM), whilst PAX5-JAK2 only responded to the JAK2 inhibitor, ruxolitinib. Notably, we show efficacy of dasatinib (20mg/kg/day p.o) in a xenograft model of ETV6-ABL1, with reduction of circulating human CD45+ cells (17.4 vs 88.2%; p These data define the genomic landscape of Ph-like ALL and show that the majority of cases harbor genetic alterations that activate a limited number of kinase signaling pathways. These results provide the basis for prospective precision medicine clinical trials that identify and direct patients with Ph-like ALL to logical TKI therapy. Citation Format: Kathryn G. Roberts, Yongjin Li, Debbie Payne-Turner, Jinghui Zhang, Richard C. Harvey, Yung-Li Yang, Guangchun Song, Jing Ma, Shann-Ching Chen, Jinjun Cheng, Natalia Santiago-Morales, Ilaria Iacobucci, Meenakshi Devidas, I-Ming Chen, Shalini Reshmi, Michael Rusch, Pankaj Gupta, Naomi J. Winick, William L. Carroll, Nyla A. Heerema, Andrew J. Carroll, Elizabeth A. Raetz, Guido Marcucci, Clara D. Bloomfield, Wendy Stock, Steven M. Kornblau, Elisabeth Paietta, Ching-Hon Pui, Sima Jeha, James Downing, Daniela S. Gerhard, Julie M. Gastier-Foster, Mignon L. Loh, Cheryl Willman, Stephen P. Hunger, Charles G. Mullighan. The genetic landscape of Ph-like acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3083. doi:10.1158/1538-7445.AM2014-3083