ALBERT

Description: Article The t(8;21) translocation is often found in acute myeloid leukaemia but is not sufficient for development of the disease. In this study, the authors identify frequent mutations in the transcriptional repressor, ZBTB7A , in these patients and show that the mutations reduce DNA binding activity. Nature Communications doi: 10.1038/ncomms11733 Authors: Luise Hartmann, Sayantanee Dutta, Sabrina Opatz, Sebastian Vosberg, Katrin Reiter, Georg Leubolt, Klaus H. Metzeler, Tobias Herold, Stefanos A. Bamopoulos, Kathrin Bräundl, Evelyn Zellmeier, Bianka Ksienzyk, Nikola P. Konstandin, Stephanie Schneider, Karl-Peter Hopfner, Alexander Graf, Stefan Krebs, Helmut Blum, Jan Moritz Middeke, Friedrich Stölzel, Christian Thiede, Stephan Wolf, Stefan K. Bohlander, Caroline Preiss, Linping Chen-Wichmann, Christian Wichmann, Maria Cristina Sauerland, Thomas Büchner, Wolfgang E. Berdel, Bernhard J. Wörmann, Jan Braess, Wolfgang Hiddemann, Karsten Spiekermann, Philipp A. Greif

Description: Background: Genome-wide association studies (GWAS) with metabolic traits and metabolome-wide association studies (MWAS) with traits of biomedical relevance are powerful tools to identify the contribution of genetic, environmental and lifestyle factors to the etiology of complex diseases. Hypothesis-free testing of ratios between all possible metabolite pairs in GWAS and MWAS has proven to be an innovative approach in the discovery of new biologically meaningful associations. The p-gain statistic was introduced as an ad-hoc measure to determine whether a ratio between two metabolite concentrations carries more information than the two corresponding metabolite concentrations alone. So far, only a rule of thumb was applied to determine the significance of the p-gain. Results: Here we explore the statistical properties of the p-gain through simulation of its density and by sampling of experimental data. We derive critical values of the p-gain for different levels of correlation between metabolite pairs and show that B/(2*alpha) is a conservative critical value for the p-gain, where alpha is the level of significance and B the number of tested metabolite pairs. Conclusions: We show that the p-gain is a well defined measure that can be used to identify statistically significant metabolite ratios in association studies and provide a conservative significance cut-off for the p-gain for use in future association studies with metabolic traits.

Description: Deregulated gene expression due to genetic alterations, such as gene fusions affecting transcription and/or epigenetic factors is the hallmark of acute myeloid leukemia and the basis for the differentiation block of hematopoietic progenitors. Acute megakaryoblastic leukemia (AMKL) is a subtype of poor prognosis acute myeloid leukemia (AML) affecting primarily young children. Recently, the ETO2-GLIS2 fusion has been identified in 20-30% of de novo AMKL and associated with the worst prognosis in this subtype of AML. To characterize the transformation induced by ETO2-GLIS2, we first defined the consequences of ETO2-GLIS2 expression on hematopoietic progenitors and the contribution of ETO2 and GLIS2 on differentiation and self-renewal. Using methylcellulose replating assays and phenotype characterization, we show that the GLIS2 moiety drives the megakaryocytic phenotype whereas both the ETO2 and GLIS2 moieties are required for maintaining self-renewal. Global expression profiling and comparison to patients' signature consistently identify ETO2-GLIS2-mediated deregulation of major transcriptional regulators of hematopoiesis and leukemogenesis, including overexpression of the ERG oncogene. ChIP-seq analysis reveals that ETO2-GLIS2 is recruited at normal ETO2 complexes sites and also at GLIS2-specific targets through binding via GLIS2 DNA-binding domain. We demonstrate that ETO2-GLIS2 fusion localize at half of H3K27Ac-dense enhancers, so called super-enhancers, to control transcription of associated genes. We show that interaction of ETO2-GLIS2 with ETO2 complexes is an essential node for the transcriptional control by the fusion at enhancer elements. Indeed, ETO2-GLIS2 dimerizes and interacts with endogenous ETO2 via its NHR2 domains. An NHR2 peptide-interference strategy inhibits oligomerization, reverses the transcriptional activation at enhancers, promotes megakaryocytic differentiation and abrogates human AMKL cells maintenance in vivo. Finally, upregulation of ERG by ETO2-GLIS2 further strengthen enhancers formation as ERG is co-recruited generating a feed forward loop at these elements and its knockdown or genetic inactivation downregulates expression of ETO2-GLIS2 targets required for leukemic cells survival. We propose that the megakaryocytic differentiation arrest and self-renewal controlled by ETO2-GLIS2 results from an imbalance in the expression of master transcription factors imposed by aberrant chromatin structures at enhancers that may be disrupted by targeting the NHR2 interface. Disclosures No relevant conflicts of interest to declare.

Description: ZBTB7A is a transcription factor involved in the regulation of metabolism and hematopoietic linage fate decisions. Recently, we found ZBTB7A mutated in 23% of Acute Myeloid Leukemia (AML) patients with t(8;21) translocation (Hartmann et al., 2016, Nat Commun). However, the oncogenic collaboration between ZBTB7A alterations and the RUNX1/RUNX1T1 fusion in AML t(8;21) remains poorly understood. To study ZBTB7A mutations in the context of RUNX1/RUNX1T1-dependent transformation, we used human CD34+ cells co-transduced with a truncated form of RUNX1/RUNX1T1 and ZBTB7A wild-type (WT) or its mutants (R402C and A175fs). We then followed the evolution of fluorescence marker positive cells over a period of 60 days. While expression of RUNX1/RUNX1T1 alone caused clonal expansion, co-expression of ZBTB7A WT impaired the outgrowth of CD34+ cells (Figure 1a). In contrast, the anti-proliferative effect of ZBTB7A was lost for both of its mutants tested resulting in a rescue of the clonal expansion (Figure 1b). To investigate the effect of ZBTB7A mutations on tumor metabolism, we used CRISPR/Cas9 to knockout (KO) ZBTB7A in the myeloid K562 cell line. As ZBTB7A is a known negative regulator of glycolysis, we treated KO and control cells with the glycolysis inhibitor 2-deoxy-d-glucouse (2-DG). KO cells were more sensitive to 2-DG compared to control cells (mean IC50: 3.06 vs 6.82 mM; p-value=0.087) (Figure 1c). These results are in line with the observed upregulation of glycolytic genes in ZBTB7A-mutant AML t(8;21) and suggest that these patients may benefit from the treatment with metabolic inhibitors. To learn more about deregulation of ZBTB7A target genes we are currently performing RNA-Seq analysis of WT vs KO K562. Moreover, we used the K562 KO model to investigate the impact of loss of ZBTB7A on myeloid differentiation. The baseline expression of the erythroid marker CD235a was reduced in KO cells. Ectopic expression of ZBTB7A WT in the KO cells restored the CD235a levels to a control level, while expression of mutants or vector showed no effect. These findings are in agreement with previous reports of ZBTB7A involvement in erythroid differentiation. To study the effect of ZBTB7A mutations on granulopoiesis, we established HL60 cells stably expressing WT or mutant ZBTB7A. We then differentiated the cells into granulocytes through all-trans retinoic acid (ATRA) treatment. Expression of WT, but not the mutants, resulted in a 4-fold increase of the granulocytic marker CD11b. Additionally, we induced monocytic differentiation through Phorbol 12-myristate 13-acetate (PMA) treatment. The mutant expressing cells showed similar levels of the monocytic marker CD14 as control cells. WT overexpressing cells had a 50% decrease in the number of monocytes. We then used CRISPR/Cas9 to establish ZBTB7A KO HL60, which exhibited a 5.5-fold increase in CD14 compared to control cells (Figure 1d). This data supports a previously unknown negative regulatory role of ZBTB7A in monocytic differentiation. With regards to potential therapeutic applications, we tested the PMA sensitivity of HL60 and found a significantly lower IC50 in absence of ZBTB7A (mean: 124.5 vs 269.8 pM; p-value=0.001). Hence, loss of ZBTB7A may facilitate the pharmacological differentiation of leukemia cells. In conclusion, ZBTB7A mutations in AML t(8;21) display a loss-of-function phenotype. Inactivating mutations of ZBTB7A allow for hCD34+ RUNX1/RUNX1T1-mediated expansion and deregulated tumor metabolism. Finally, loss of ZBTB7A expression perturbs myeloid development and thus may complement the block of differentiation induced by RUNX1/RUNX1T1. These findings open up avenues to novel therapies for ZBTB7A mutated patients including metabolic inhibition and pharmacological differentiation. Disclosures Hiddemann: F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: The translocation t(8;21) is the most common chromosomal rearrangement in AML and generates the RUNX1/ETO fusion protein. RUNX1/ETO is required for maintaining the leukaemic phenotype influencing both leukaemic clonogenicity and proliferation and is traditionally thought of as a transcriptional repressor through its actions with histone deacetylases and transcriptional co-repressors. Intriguingly our RNAi experiments have also identified many RUNX1/ETO target genes that are transcriptionally upregulated by this fusion protein. To delve deeper into the involvement of these upregulated genes in RUNX1/ETO driven leukaemia, targeted RNAi screens were performed both in vitro and in vivo, directed at genes found directly bound by RUNX1/ETO and that were found differentially expressed upon RUNX1/ETO knockdown. For the RNAi screens, we used a doxycycline (dox)-inducible lentiviral RNAi library covering each gene with 3 shRNAs. We transduced two t(8;21)-positive AML cell lines, Kasumi-1 and SKNO-1, with this library and performed parallel screens employing colony formation and long-term suspension culture assays in the in vitro arm, and intrafemoral xenotransplantation of immunodeficient NSG mice for the in vivo arm. For comparative purposes, both arms of the RNAi screen were performed in the presence and absence of dox. DNA was isolated throughout both screens and was analysed by Next Generation Sequencing (NGS). Comparison of the changes in level of integrated shRNA coding sequences between dox and no dox groups were made using DESEQ and those genes that are required for RUNX1/ETO driven leukaemia identified. As expected, RUNX1/ETO shRNA constructs diminished upon induction of shRUNX1/ETO expression by dox, therefore implicating RUNX1/ETO dependency in cell survival. Furthermore non-targeting control shRNA (shNTC) levels were unaffected. Out of the many hits identified (which include previously reported hits such as Pontin (RUBVL1), SKP2 and KIT, Cyclin D2 (CCND2) stood out significantly, since levels of shCCND2 were depleted in all dox samples from both arms of the screen. CCND2 is a cell cycle regulator whose activity is dependent on its binding to CDK4/6 in G1 phase. Phosphorylation of Rb (Retinoblastoma), by CDK4/6-CCND2, uncouples Rb from E2F allowing transcription of essential S phase genes. Here we show CCND2 knockdown downregulates both cell proliferation and colony formation in t(8;21) positive cells by causing G1 phase cell cycle arrest via a reduction in Rb phosphorylation, which is a phenotype copied by our RUNX1/ETO knockdown. Moreover, inhibition of CDK4/6-CCND2 by palbociclib (PD-0332991) in t(8;21) positive cells similarly reduces cell proliferation and colony formation via a G1 cell cycle arrest. Interestingly, primary CD34+ human stem/progenitor cells (HSPCs) containing an activated form of KIT (N822K) and the RUNX1/ETO 9a isoform are likewise responsive to palbociclib treatment. In addition to in vitro studies, palbociclib was tested in vivo in two separate experimental models: firstly, mice were intravenously injected with murine CD34+ cKit+ HSPCs expressing the RUNX1/ETO9a isoform and secondly, immunodeficient mice were intrahepatically injected with Kasumi-1. Mice were dosed with palbociclib upon confirmation of engraftment. Engraftment was detected using bioluminescence and survival rates recorded. Both studies showed significant increased survival with palbociclib treatment with the first giving an increased median survival of 59 versus 29 days (p

Description: The transcription factor Stat5 mediates the cellular response to activation of multiple cytokine receptors involved in the regulation of proliferation and differentiation of hematopoietic cells. Recently, the human Stat5 gene was found to be translocated to the RARα gene in a patient with acute promyelocytic leukemia indicating that Stat5 might also play a role in cellular transformation. We investigated the mechanism by which Stat5 might exert this function and studied the biochemical and cellular functions of fusion proteins comprising Stat5 and RARα. The expression of Stat5-RARα causes the transcriptional repression of gene transcription, a process that requires the coiled-coil domain of Stat5 (amino acid positions 133-333). Oligomerization of this domain in the Stat5-RARα fusion protein leads to stable binding of the corepressor SMRT independent of all-trans retinoic acid (ATRA) stimulation and is accompanied by an impaired response to differentiation signals in hematopoietic cells. This inhibitory effect on myeloid differentiation cannot be overcome by simultaneous coexpression of RARα. We conclude that Stat5 is capable of interacting with a corepressor complex that alters the pattern of corepressor binding to RARα and its dissociation in response to ATRA stimulation, leading to enhanced repressor activity and a block of hematopoietic differentiation.

Description: The transcription factor GATA2 plays an important role in cell lineage decisions during hematopoiesis. GATA2 Zinc-Finger (ZF) mutations are associated with distinct entities of myeloid malignancies. Alterations of the N-terminal ZF1 were identified in AML patients with biallelic CEBPA mutations, whereas the C-terminal ZF2 is typically affected by germline mutations predisposing to MDS and AML, or by somatic lesions in CML blast crisis. Nevertheless, the context-dependent mechanisms underlying GATA2 ZF mutations remain mostly unclear. Here, we set out to study the functional consequences of GATA2 ZF mutations. To test the effect on differentiation, we expressed GATA2 wild-type (WT) or GATA2 ZF mutants in human CD34+ hematopoietic stem and progenitor cells, stimulated with appropriate cytokines. Differentiation was evaluated by FACS-measurements of surface marker expression (erythroid markers: CD71, CD235a; granulocytic/monocytic markers: CD15, CD14). GATA2 WT caused a block of erythroid differentiation that is overcome by the ZF1 mutants (A318T and G320D), whereas the ZF2 mutant L359V may aggravate this block. For granulocytic/monocytic differentiation an overall block was observed for GATA2 WT and all the ZF mutants tested (Figure 1 A, B). Recently, we and others observed GATA2 mutation gain in AML relapse (Greif et al., 2018 Clin Cancer Res; Christopher et al., 2018, NEJM), pointing towards a potential role in therapy resistance. Therefore, we treated K562 cells stably expressing GATA2 WT or mutants with Daunorubicin (one of the two drugs commonly used in AML chemotherapy). Expression of GATA2 A318T in K562 cells correlated with higher sensitivity to Daunorubicin and lower expression levels of IDH2. (Figure 1 C, D). Interestingly, this particular ZF1 mutation was the only one that got lost at relapse in the study by Christopher and colleagues, consistent with therapy sensitivity, whereas most of the GATA2 mutations gained at relapse were localized in the ZF2 domain. In summary, GATA2 ZF mutations influence hematopoietic differentiation and chemotherapy response in a position-dependent manner. In the present study, we report distinct roles for individual GATA2 mutations depending on the affected ZF domain and altered amino acid positions. Understanding the oncogenic collaboration of GATA2 mutations with other driver genes in distinct patient subgroups is a challenge ahead. Figure.1 Disclosures Hiddemann: Roche: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria; Bayer: Research Funding; Vector Therapeutics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding.

Description: About 12% of all de novo acute myeloid leukemias are characterized by the translocation t(8;21) which generates the oncogenic fusion protein AML1/ETO. AML1/ETO is found in high molecular weight complexes (HMWC) which are crucial for the block in myeloid differentiation of AML1/ETO transformed cells. Essential for HMWC formation is the alpha-helical nervy homology region 2 (NHR2) within ETO which serves as an interacting surface for oligomerization as well as association with members of the ETO protein family. Based on the observation that the integrity of the NHR2 domain is essential for transcriptional repressor activity of ETO, we designed a peptide (NC128) aimed to selectively interfere with AML1/ETO oligomerization. Confocal laser scanning microscopy revealed colocalization of NC128 with AML1/ETO in the nucleus. In protein-protein interaction studies we could demonstrate that the NHR2 domain in NC128 was necessary and sufficient for proper binding to AML1/ETO. Expression of NC128 in AML1/ETO positive cells led to inhibition of AML1/ETO HMWC formation as demonstrated by size-exclusion chromatography. NC128 expression in the AML1/ETO growth dependent Kasumi-1 cells restores transcription of AML1/ETO target genes. Among others, the expression of PU.1, a master regulator of myeloid differentiation, was significantly upregulated in the presence of NC128. In Kasumi-1 cells as well as in U937-AML1/ETO cells expression of NC128 almost completely overcomes the block of cytokine mediated differentiation induced by AML1/ETO. In the presence of NC128, Kasumi-1 cells lose their progenitor cell characteristics and upregulate marker of early monocytic differentiation. Likewise, NC128 expressing Kasumi-1 and SKNO-1 cells are arrested in cell cycle progression with a peak in the G1 phase of the cell cycle. The binding affinity of NHR2 peptide mutants to ETO directly correlates with the intensity of growth inhibition. A derivative of NC128 which retained all NHR2 amino acids (N89) maintained full binding capacity to ETO as well as antiproliferative effects, whereas a mutant of N89 lacking 7 C-terminal amino acids (N82) significantly lost binding capacity and its antiproliferative effect. Next a codon optimized expression construct was developed in order to increase the cellular expression levels of N89. Compared to N89, expression of this construct enhances growth arrest suggesting that the NHR2 peptides act in a dose dependent manner. To investigate the influence of NC128 on primary cells, human CD34 progenitor cells were immortalized with AML1/ETO. Expression of NC128 in these cells resulted in growth arrest, loss of CD34 expression and an increased rate of apoptosis. In order to directly deliver the peptide to AML1/ETO expressing cells, the NHR2 domain was fused to the HIV-1 Tat protein transduction domain. The bacterial expressed peptides were purified by affinity chromatography and were proven to be stable in serum-containing medium for several hours. Upon protein transduction into mammalian cells, recombinant NHR2 peptides could be detected in cellular lysates. Furthermore, we already can show by co-immunoprecipitation experiments that the transducible peptides are able to specifically interact with ETO protein. Our results propose that selective interference with NHR2-mediated oligomerization could provide a promising strategy for the inhibition of the oncogenic properties of AML1/ETO. Based on our results, we are aiming to develop screening strategies for both peptides and small molecule substances to interfere with NHR2 mediated AML1/ETO complex formation.

Description: RUNX1/ETO, the fusion protein resulting from the chromosomal translocation t(8;21), is one of the most frequent translocation products in acute myeloid leukemia. Several in vitro and in vivo studies have shown that the homo-tetramerization domain of ETO, the nervy homology region 2 (NHR2), is essential for RUNX1/ETO oncogenic activity. We analyzed the energetic contribution of individual amino acids within the NHR2 to RUNX1/ETO dimer-tetramer transition and found a clustered area of 5 distinct amino acids with strong contribution to the stability of tetramers. Substitution of these amino acids abolishes tetramer formation without affecting dimer formation. Similar to RUNX1/ETO monomers, dimers failed to bind efficiently to DNA and to alter expression of RUNX1-dependent genes. RUNX1/ETO dimers do not block myeloid differentiation, are unable to enhance the self-renewal capacity of hematopoietic progenitors, and fail to induce leukemia in a murine transplantation model. Our data reveal the existence of an essential structural motif (hot spot) at the NHR2 dimer-tetramer interface, suitable for a molecular intervention in t(8;21) leukemias.