ALBERT

Description: Homeobox genes (HOX) encode transcription factors that are frequently deregulated in leukemias. Our previous findings described that HOX gene expression differs among genetically characterized subtypes of pediatric AML with PML-RARa+ patients having the lowest overall HOX gene expression. We observed that HOX gene expression positively correlated with expression of histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX and negatively with DNA methyltransferase DNMT3b. Interestingly, it has been shown that JMJD3 is a direct target of PML-RARa protein (Martens, JH et al, 2010, Cancer Cell). These findings led us to postulate the hypothesis that reduced levels of HOX genes in PML-RARa+ AML can be caused by the suppressed expression of histone demethylases, such as JMJD3 and UTX, resulting in increased H3K27 methylation and transcription inhibition. We chose PML-RARa+ NB4 cell line to study the role of PML-RARa fusion gene in the regulation of HOX gene expression. To inhibit the effect of PML-RARa we used all-trans retinoic acid (ATRA; 1 uM, 10 uM) which was described to release the block caused by this fusion protein. Expression of particular HOX genes (e.g., HOXA1, HOXA3, HOXA5, HOXA7) together with that of JMJD3 and UTX assessed by qPCR was significantly elevated after ATRA treatment, while gene expression of DNMT3b was decreased. To test whether the reduction in HOX gene expression is directly related to the levels of JMJD3 and UTX, we cultured NB4 cells with a specific inhibitor of these histone demethylases, GSK-J4 (1 uM, 10 uM), in combination with ATRA. This co-treatment led to inhibition of JMJD3 and UTX proteins, followed by significant reduction of HOX genes expression (e.g., HOXA1, HOXA3, HOXA5, HOXA7). This result supports our hypothesis that HOX genes expression is directly related to JMJD3/UTX activity. To determine the effect of ATRA and GSK-J4 on histone marks we have isolated histones by acid extraction and detected the levels of histones by western blot in NB4 ATRA or GSK-J4/ATRA treated cells. We observed that the level of repressive histone methylation mark (trimethylated H3K27; H3K27me3) was decreased after ATRA treatment (activation of JMJD3/UTX) and increased after GSK-J4/ATRA co-treatment (inhibition of JMJD3/UTX). The opposite effect was observed in active histone methylation marks where di- and tri-methylated H3K4 (H3K4me2, H3K4me3) increased after ATRA treatment and decreased after GSK-J4/ATRA co-treatment. H3K9 dimethylated (another repressive histone methylation mark) levels did not change. Next, to investigate the histone code directly in particular HOX genes regions we performed chromatin immunoprecipitation (ChIP) assays. We studied the presence of H3K27me3 and H3K4me2 in 5´UTR genomic region of particular HOX genes (HOXA1, HOXA2, HOXA3, HOXA5, HOXA7) in cells treated with ATRA alone or in the combination with GSK-J4. Preliminary results showed reduction in repressive marks (H3K27me3) upon ATRA treatment, whereas addition of GSK-J4 prevented this decrease. Accordingly, we observed that ATRA/GSK-J4 co-treatment reduced active histone mark H3K4me2. To evaluate the role of DNA methylation in observed expression changes after ATRA treatment we performed bisulfite sequencing of particular promoter sites of HOX genes (e.g., HOXA7, HOXA5). Although we detected decreased DNMT3b gene expression after ATRA treatment there was no change in DNA methylation of CpGs in studied regions. Our results demonstrate that changes in chromatin activity correspond with changes in HOX gene expression. Moreover, ChIP data show direct binding of the modified histones and HOX 5´UTR sites. Our data implicate histone demethylases in regulation of HOX gene expression in PML-RARa+ leukemic blasts. DNA methylation in these particular HOX genes is not involved in the regulation. Elucidating the mechanism of regulation of HOX genes expression can help to understand their role in the leukemogenic process. Supported by GACR P304/12/2214 and GAUK 568213. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 779 L-asparaginase (L-asp) is an important component of childhood acute lymphoblastic leukemia (ALL) therapy. Its cytotoxic effect is based on the depletion of extracellular asparagine and glutamine. Leukemic cells are sensitive to this depletion due to the lower activity of asparagine synthetase compared to healthy cells. However, the mechanism of resistance development remains unclear. The aim of this study was to obtain further insights into the mechanisms underlying the cytotoxic effect of L-asp. We used two models: resistant preB ALL leukemic cells derived from the REH (TEL/AML1[+]; very sensitive) and NALM6 (TEL/PDGFRB[+]; medium sensitive) cell lines by long-term incubation with L-asp. As a second model we used REH and NALM6 incubated with L-asp for 24 hrs, mimicking acute phase of treatment. We performed GEP of REH, res-REH, NALM6 and res-NALM6. There was an overlap of 30 genes changed in both cases. Applying the pathway analysis we found that L-asp influences the regulation of lipid metabolism and apoptosis regulated by mitochondrial proteins. Next we merged our data with GEP data published by Holleman et al. (NEJM, 2004) of ALL patients′ samples sensitive/resistant to L-Asp. Two pathways, one regulating protein translation, the other metabolism, scored very highly. Next to glucose, glutamine is the other major cellular energy source, it is also important for activation of PI3K/Akt/mTOR pathway - the key regulator of translation. Since L-asp also depletes glutamine, we focused on the effect of L-asp treatment on bioenergetics and translation in leukemic cells. To confirm the effect of L-asp on PI3K/Akt/mTOR pathway we measured the amount of Akt protein and P70S6K/p-P70S6K (downstream target of mTOR) in REH and NALM6 treated for 24 hours with L-asp. Akt and p-P70S6K expression fell in both cell lines. Next we were interested if PI3K/mTOR inhibition affects the sensitivity of resistant cell lines. We measured the cytostatic effect of co-treatment: L-asp with mTORC1 inhibitor (rapamycin); with dual inhibitor of mTORC1 and PI3K (LY294002); and with specific inhibitor of PI3K (PX866) on REH, res-REH, NALM6 and res-NALM6. The MTS assay proved the L-asp/rapamycin combination the most effective. It strongly diminished viability of all cell lines, even in those resistant to L-asp. c-Myc as an activator of glutamine catabolism showed a significant decrease in REH and NALM6 after incubation with L-asp. In addition, c-Myc was also decreased in res-REH compared with REH. Importantly, c-Myc activates glycolysis which is a main energy generator in cancer cells rather than oxidative phosphorylation (OXPHOS). We showed diminishement of Glucose transporter type 1 protein expression and 2.5-times reduced level of lactate, the product of cancer cell glycolysis, in media after L-asp exposure. Next we measured cell respiration that mirrors oxidative fosforylation (OXPHOS). REH and NALM6 showed increased capacity of respiratory chain after L-asp exposure, which was observed as an increase of endogenous respiration in uncoupled state. REH boosted the capacity of the respiratory chain from 163 to 236 pmol O2/s/mg (p

Description: Introduction: Recently we described a subgroup of pediatric patients with B cell precursor acute lymphoblastic leukemia (BCP ALL) with switching from B to monocytic lineage in early phase of the therapy (Slamova et al., 2014). In a limited cohort of patients with switching ALL (swALL), we observed inferior response to treatment with discrepancy of minimal residual disease level (MRD) assessed by flow cytometry (FC) and quantitative polymerase chain reaction (qPCR) of Immunoglobulin-T cell receptor (Ig-TCR) rearrangements. In current Berlin-Frankfurt-Münster (BFM) treatment protocols, FC MRD value at day 15 (d15) and PCR MRD value at day 33 (d33) and week 12 (w12) are used for stratification. Using an extended cohort of patients with available RNA sequencing data (cohort mainly focused on B other cases or swALLs) we aimed to answer following questions:What is the genetic background of swALL? What is the frequency among swALLs of the recently described DUX4 rearranged subgroup?How do B cell oriented FC and PCR MRD correlate in standard protocol timepoints, i.e. day 8 (d8) (peripheral blood, PB) d15, d33 and w12 (bone marrow, BM) of treatment?What is the characteristic MRD response to treatment in swALL? Results:We performed RNA sequencing in 177 patients (median age 6.1 years, range 0-18) treated by several treatment protocols (ALL BFM 95 n=5, ALL IC BFM 2002 n=14, ALL AIEOP BFM 2000 n=17, ALL AIEOP BFM 2009 n=135, Interfant n=3, ALL IC/Interfant n=2, EsPhALL n=1). In 68 patients we observed switching phenomenon by appearance of B/monocytoid population coexpressing B lineage (CD19, CD34) and monocytic lineage (CD33, CD14) markers (median 0.98%, range 0.032-38%). In non swALLs median of this population was 0.059% (range 0.0025-1.1%) and the cells did not form a clear cluster. According to RNAseq data, majority of swALL patients (n=42/68) belong to DUX4 subgroup (chi square p〈 0.00001). The distribution into other molecular genetic subtypes is summarized in table 1.Correlation coefficient (Spearman) of all included samples with both available values (n=552) was 0.82 (p

Description: Acute lymphoblastic leukemia (ALL) is the most frequent type of childhood cancer. The key component in the therapy, L-asparaginase (ASNase), hydrolyzes plasma asparagine and glutamine. Leukemic cells are sensitive to the depletion due to low activity of asparagine synthetase. Although the treatment is very effective, resistance and side effects remain a serious problem in some cases and its mechanism of action is not well understood. Our aim is to clarify the intracellular consequences of the amino acid depletion to define the reason of different patients´ response. We have generated ASNase-resistant subclones through chronic exposure to the enzyme. Pathway analysis of gene expression profiles of the cell lines (REH;TEL/AML1-positive, NALM-6; TEL/PDGFRB1-positive and their resistant counterparts) and primary samples (sensitive and resistant to ASNase; Holleman et al. (NEJM, 2004)) revealed that ASNase affects the translation machinery and metabolism of leukemic cells. The key nutrient sensor positively regulating protein synthesis, pyrimidine synthesis, glycolysis and lipid synthesis is mTORC1. Since ASNase depletes glutamine that is essential for mTORC1 activity, we hypothesized that the effect of ASNase is driven through mTORC1 signaling. Main aim of the study was to explore the effect of ASNase on downstream mTORC1 targets in leukemic cells. ASNase treatment inhibited protein synthesis, displayed by dephosphorylation of p-P70SK6 and p-S6. ASNase also decreased de-novo DNA synthesis as shown by dephosphorylation of p-CAD. This result was confirmed by analysis of de-novo pyrimidine synthesis intermediates, uridine monophosphate and uridine, measured by UPLC-ToF-MS. Both were decreased upon ASNase treatment. Except experiments done on ALL cell lines we also detected dephosphorylation of p-S6, p-CAD in primary samples. Regarding the effect on glycolysis we observed inhibition of glucose uptake and decrease of lactate production in cells treated with ASNase. We also detected the decrease of protein levels of c-Myc, the activator of glucose and glutamine catabolism, and glucose transporter 1 (Glut-1). On the contrary, ASNase increased fatty acid oxidation (FAO) followed by the elevation of the capacity of cell respiration and NAD+/NADH ratio in both cell lines. Next, we wanted to elucidate whether ASNase inhibits mTORC1 targets through RagB, the key protein mediating response to general amino acid deprivation. We established a RagB mutant leukemic cell line with constitutive activation of mTORC1 pathway despite deprivation of amino acids. ASNase treatment did not inhibit p-S6 and p-CAD in this cell line. Similarly, ASNase failed to increase FAO in cells with active mTORC1. These results suggest that the effect of ASNase on protein translation, de novo pyrimidine synthesis and FAO is mediated through RagB-mTORC1 pathway. By contrast, c-Myc expression was decreased in both RagB wild type and mutant cells, indicating that ASNase inhibits glycolysis in RagB-mTORC1 independent manner. The activation of FAO has been suggested to have a pro-survival function in leukemic cells under nutrient stress conditions. We tested whether the increase of FAO in ALL cells treated with ASNase also serves to cope with the metabolic stress. Pharmacological inhibition of FAO significantly increased the sensitivity of ALL cells to ASNase. Moreover, cells with the inability to increase FAO (RagB mutant) were more sensitive to ASNase compared to RagB wild type cells. Our results show that the inhibitory effect of ASNase on mTORC1 leads not only to apoptosis but also to metabolic reprogramming. We propose that inhibition of protein translation and pyrimidine synthesis are part of apoptotic processes whereas increased FAO and cell respiration represent pro-survival pathway. Altogether, our study suggests that targeting of FAO in leukemic cells resistant to ASNase is a promising new therapeutic strategy. IGA-NT1249, GAUK-632513 Disclosures No relevant conflicts of interest to declare.

Description: Leukemia is a complex disease pathologically manifested at the DNA, mRNA and protein level. Understanding leukemia pathogenesis is prevalently focused on mutations at the DNA (or mRNA) level, however the functional consequences of these changes on cellular machineries are not fully clarified. Since proteome analysis provides link between gene sequence and cellular physiology, proteomics can contribute to elucidate mechanism of disease and response to treatment. Moreover some alterations are manifested only at the protein level including subcellular localisation, post-translational modification (e.g. phosphorylation), protein cleavage or protein-protein interactions. Performing large-scale protein analysis of primary leukemia samples requires the development of more effective proteomic approaches as well as new analytical strategies. Here, we present novel microsphere-based antibody array format with automatical analysis tool that can follow changes in expression and post-translational modification of leukemia associated proteins with regards to intracellular localisation and protein cleavage in primary childhood acute leukemia (AL). Size Exclusion Chromatography-Microsphere-based Affinity Proteomics (SEC-MAP) is a set of 1728 populations of fluorescently-labeled microbeads, each carrying an antibody against respective human protein. Native cellular proteins (and their complexes) are isolated using detergents, labeled with biotin and subjected to size exclusion chromatography to obtain 24 molecular weight fractions. The fractions are incubated with SEC-MAP microbeads and the antibody-protein binding is detected using fluorescently-labeled streptavidin by flow cytometry. Flow cytometer resolves color-code of each microbead population and reads the amount of bound protein. The signals from 24 size fractions are combined and protein binding is detected as protein reactivity peaks similar to bands on western blot. The analysis is performed using automatic software created in R. It allows for automatic processing of fcs files as well as advanced follow-up analysis including quality control, normalisation, protein peaks recognition and clustering of results We have examined the expression of cytoplasmic (n=980) and membrane (n=769) proteins in 69 primary samples of AL obtained at diagnosis according to the Institutional Ethics Committee Giudelines. For the normalisation of protein expression we have used Loess normalisation commonly used in mRNA profiling studies. Due to ability of SEC-MAP to separate proteins according their molecular weight we have identified not only the expression of proteins but also the size that corresponds to its monomeric or multimeric presence and furthermore could serve as a control of proteolysis. We have revealed the sensitivity to proteolysis of 4 standard house-keeping proteins (Akt, Abl, β-actin and β2-microglobulin). Abl and Akt proved to be better controls of proteolysis. Detected with SEC-MAP or western blot, β-actin and β2-microglobulin, unlike Abl and Akt, have not been found in their cleaved forms in the proteolytically digested samples. Thus we have identified proteolysis in 12 samples which have been subsequently excluded from the analysis. So far we have identified 44 proteins (including CD markers distinguishing lineage specificity e.g. CD22, CD3, CD33) which have been differentially expressed in different subtypes of AL (B-cell precursor acute lymphoblastic leukemia, BCP-ALL, n=35), T-cell acute lymphoblastic leukemia (T-ALL, n=9) and acute myeloid leukemia (AML, n=13) (Multiple Testing Procedures - Bioconductor Package multtest, p

Description: Flow cytometry is an important tool both for research and diagnostics of hematologic malignancies - including monitoring of minimal residual disease (MRD). Recent progress and more widespread availability of 6 and higher color flow cytometry leads to complex, information-rich datasets which are very challenging to analyze. Here, we validate a novel approach to multi-parameter flow data analysis in MRD flow data sets from 39 ALL patients (including 23 patients from the I-BFM list mode data (LMD) ring trial). The approach combines hierarchical clustering (HCA) using a newly developed algorithm and support vector machine (SVM) learning. The algorithm employs a scale-invariant Mahalanobis distance measurement for merging clusters. This reflects the extended ellipsoid shape of the populations and is better suited for flow cytometric data compared with standard HCA metrics. The resulting hierarchical tree, combined with the heatmap of the CD marker expression allows visualization of hierarchically clustered data of all analyzed parameters displayed in a single plot. The clusters from HCA (representing the ALL blast population at diagnosis) were used to train SVM classifiers which were then applied to test for presence of a matching population in the test sample (follow-up sample). All work was carried out in MATLAB (MathWorks, Inc.). Using HCA, we have been able to detect the leukemic blast population in diagnostic and follow-up datasets (n=81) from three centers. The correlation (Pearson correlation coefficient = 0.98) between HCA and the standard gating approach was highly comparable to inter-laboratory comparisons within the I-BFM LMD ring trial (Dworzak MN et al; Cytometry B Clin Cytom. 2008 Jun 11.). To further improve sensitivity and exact quantification of low MRD levels and to automate MRD detection, we combined HCA with SVM learning. We have analyzed 21 samples from 5 patients with MRD levels between 0.004 to 57.54%. HCA plus SVM correlated better with standard gating results than HCA alone in particular in samples with low MRD levels (

Description: Summary ShinySOM offers a user-friendly interface for reproducible, high-throughput analysis of high-dimensional flow and mass cytometry data guided by self-organizing maps. The software implements a FlowSOM-style workflow, with improvements in performance, visualizations and data dissection possibilities. The outputs of the analysis include precise statistical information about the dissected samples, and R-compatible metadata useful for the batch processing of large sample volumes. Availability and implementation ShinySOM is free and open-source, available online at gitlab.com/exaexa/ShinySOM. Supplementary information Supplementary data are available at Bioinformatics online.