Jasmin Corso

RUNX1 represses the erythroid gene expression program during megakaryocytic differentiation.

The activity of antagonizing transcription factors represents a mechanistic paradigm of bidirectional lineage-fate control during hematopoiesis. At the megakaryocytic/erythroid bifurcation, the cross-antagonism of krueppel-like factor 1 (KLF1) and friend leukemia integration 1 (FLI1) has such a decisive role. However, how this antagonism is resolved during lineage specification is poorly understood. We found that runt-related transcription factor 1 (RUNX1) inhibits erythroid differentiation of murine megakaryocytic/erythroid progenitors and primary human CD34(+) progenitor cells. We show that RUNX1 represses the erythroid gene expression program during megakaryocytic differentiation by epigenetic repression of the erythroid master regulator KLF1. RUNX1 binding to the KLF1 locus is increased during megakaryocytic differentiation and counterbalances the activating role of T-cell acute lymphocytic leukemia 1 (TAL1). We found that corepressor recruitment by RUNX1 contributes to a block of the KLF1-dependent erythroid gene expression program. Our data indicate that the repressive function of RUNX1 influences the balance between erythroid and megakaryocytic differentiation by shifting the balance between KLF1 and FLI1 in the direction of FLI1. Taken together, we show that RUNX1 is a key player within a network of transcription factors that represses the erythroid gene expression program.

β2 integrin–derived signals induce cell survival and proliferation of AML blasts by activating a Syk/STAT signaling axis

Spleen tyrosine kinase (Syk) induces cell survival and proliferation in a high proportion of acute myeloid leukemia (AML) blasts, but the underlying molecular events of Syk signaling have not been investigated. Proteomic techniques have allowed us to identify the multiprotein complex that is nucleated by constitutively active Syk in AML cells. This complex differs from the B-lymphoid Syk interactome with respect to several proteins, especially the integrin receptor Mac-1, the Fc-γ receptor I (FcγRI), and the transcription factors STAT3 and STAT5. We show in several AML cell line models that tonic signals derived from the Fc-γ chain lead to Syk-dependent activation of STAT3 and STAT5, which in turn induces cell survival and proliferation. Moreover, stimulation of Mac-1 or FcγRI intensifies the constitutive Syk-mediated STAT3/5 activation in AML cells, a scenario likely to take place in the bone marrow niche. In accordance with these findings, we observed that β2 integrins, including Mac-1, trigger proliferation of AML cells in an AML cell/stroma coculture model. Taken together, we identified an oncogenic integrin/Syk/STAT3/5 signaling axis that might serve as a therapeutic target of AML in the future.

FLT3-ITD and TLR9 use Bruton tyrosine kinase to activate distinct transcriptional programs mediating AML cell survival and proliferation

Acute myeloid leukemia (AML) is driven by niche-derived and cell-autonomous stimuli. Although many cell-autonomous disease drivers are known, niche-dependent signaling in the context of the genetic disease heterogeneity has been difficult to investigate. Here, we analyzed the role of Bruton tyrosine kinase (BTK) in AML. BTK was frequently expressed, and its inhibition strongly impaired the proliferation and survival of AML cells also in the presence of bone marrow stroma. By interactome analysis, (phospho)proteomics, and transcriptome sequencing, we characterized BTK signaling networks. We show that BTK-dependent signaling is highly context dependent. In Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD)-positive AML, BTK mediates FLT3-ITD-dependent Myc and STAT5 activation, and combined targeting of FLT3-ITD and BTK showed additive effects. In Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD)-negative AML, BTK couples Toll-like receptor 9 (TLR9) activation to nuclear factor κΒ and STAT5. Both BTK-dependent transcriptional programs were relevant for cell cycle progression and apoptosis regulation. Thus, we identify context-dependent oncogenic driver events that may guide subtype-specific treatment strategies and, for the first time, point to a role of TLR9 in AML. Clinical evaluation of BTK inhibitors in AML seems warranted.

Hoxa9 and Meis1 cooperatively induce addiction to syk signaling by suppressing miR-146a in acute myeloid leukemia

Summary The transcription factor Meis1 drives myeloid leukemogenesis in the context of Hox gene overexpression but is currently considered undruggable. We therefore investigated whether myeloid progenitor cells transformed by Hoxa9 and Meis1 become addicted to targetable signaling pathways. A comprehensive (phospho)proteomic analysis revealed that Meis1 increased Syk protein expression and activity. Syk upregulation occurs through a Meis1-dependent feedback loop. By dissecting this loop, we show that Syk is a direct target of miR-146a, whose expression is indirectly regulated by Meis1 through the transcription factor PU.1. In the context of Hoxa9 overexpression, Syk signaling induces Meis1, recapitulating several leukemogenic features of Hoxa9/Meis1-driven leukemia. Finally, Syk inhibition disrupts the identified regulatory loop, prolonging survival of mice with Hoxa9/Meis1-driven leukemia.

Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

Significance B-cell receptor (BCR) signaling promotes the survival of malignant B cells, such as Burkitt’s lymphoma (BL) and the activated B-cell–like subtype of diffuse large B-cell lymphoma (ABC-DLBCL). In contrast to ABC-DLBCL, which depends on chronic activation of the BCR, BL cells rely on tonic BCR signaling that is antigen-independent. Elucidation and systematic comparison of tonic and activated BCR signaling led to the identification of novel signaling effectors, including ACTN4 and ARFGEF2, which were identified as regulators of BL-cell survival. Beyond its relevance to the understanding of BL pathogenesis and the development of targeted therapies, our study complements the general understanding of BCR-induced processes also in physiological settings. Burkitts lymphoma (BL) is a highly proliferative B-cell neoplasm and is treated with intensive chemotherapy that, because of its toxicity, is often not suitable for the elderly or for patients with endemic BL in developing countries. BL cell survival relies on signals transduced by B-cell antigen receptors (BCRs). However, tonic as well as activated BCR signaling networks and their relevance for targeted therapies in BL remain elusive. We have systematically characterized and compared tonic and activated BCR signaling in BL by quantitative phosphoproteomics to identify novel BCR effectors and potential drug targets. We identified and quantified ∼16,000 phospho-sites in BL cells. Among these sites, 909 were related to tonic BCR signaling, whereas 984 phospho-sites were regulated upon BCR engagement. The majority of the identified BCR signaling effectors have not been described in the context of B cells or lymphomas yet. Most of these newly identified BCR effectors are predicted to be involved in the regulation of kinases, transcription, and cytoskeleton dynamics. Although tonic and activated BCR signaling shared a considerable number of effector proteins, we identified distinct phosphorylation events in tonic BCR signaling. We investigated the functional relevance of some newly identified BCR effectors and show that ACTN4 and ARFGEF2, which have been described as regulators of membrane-trafficking and cytoskeleton-related processes, respectively, are crucial for BL cell survival. Thus, this study provides a comprehensive dataset for tonic and activated BCR signaling and identifies effector proteins that may be relevant for BL cell survival and thus may help to develop new BL treatments.

Modulation of TCR responsiveness by the Grb2-family adaptor, Gads.

T cell antigen receptor (TCR) signaling depends on three interacting adaptor proteins: SLP-76, Gads, and LAT. Their mechanisms of signaling have been extensively explored, with the aid of fortuitously isolated LAT- and SLP-76-deficient T cell lines, but no such tools were available for Gads, a Grb2-family adaptor that bridges the TCR-inducible interaction between SLP-76 and LAT. TALEN-directed genome editing was applied to disrupt the first coding exon of human Gads in the Jurkat T cell line. Gads was dispensable for TCR-induced phosphorylation of SLP-76, but was a dose-dependent amplifier of TCR-induced CD69 expression. Gads conferred responsiveness to weak TCR stimuli, leading to PLC-γ1 phosphorylation and calcium flux. TALEN-derived, Gads-deficient T cell lines provide a uniquely tractable genetic platform for exploring its regulatory features, such as Gads phosphorylation at T262, which we observed by mass spectrometry. Upon mutation of this site, TCR responsiveness and sensitivity to weak TCR stimuli were increased. This study demonstrates the feasibility of TALEN-based reverse genetics in Jurkat T cells, while enriching our understanding of Gads as a regulated modulator of TCR sensitivity.

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

Syk and Btk induce cell survival and proliferation in AML cells by activating distinct signaling pathways and transcriptional programs.

gutachter Bernd Alt-Epping, Reinhard Andreesen, Elisabeth Andritsch, Anne Angelillo-Scherrer, Dirk Arnold, Renate Arnold, Ulrike Bacher, Stefan Balabanov, Marija Balic, Mario Bargetzi, Hannsjörg Baum, Walter Baumann, Dietrich W. Beelen, Lothar Bergmann, Jörg Beyer, Mascha Binder, Carsten Bokemeyer, Peter Borchmann, Martin Bornhäuser, Thomas Brodowicz, Peter Brossart, Tim Brümmendorf, Christian Buske, Richard Cathomas, Maximilian Christopeit, Maria De Santis, Maike De Wit, Christine Dierks, Uta Dirksen, Johannes Drach, Martin Dreyling, Ulrich Dührsen, Justus Duyster, Alexander Egle, Hermann Einsele, Wolfgang Eisterer, Jutta Engel, Monika Engelhardt, Andreas Engert, Jonas Walter Feilchenfeldt, Walter Fiedler, Thomas Fischer, Michael Fridrik, Martin Früh, Reinhard Gager, Simon Gampenrieder, Arnold Ganser, Günther Gastl, Armin Gerger, Ulrich Germing, Michael Girschikofsky, Heinz Gisslinger, Hartmut Goldschmidt, Hildegard Greinix, Viktor Grünwald, Cornelie Haag, Rainer Haas, Torsten Haferlach, Michael Herold, Viviane Hess, Pia Heußner, Wolfgang Hiddemann, Andreas Hochhaus, Friedemann Honecker, Yvonne Hummel, Dirk Jäger, Karin Jordan, Christian Junghanß, Ursula Kapp, Felix Keil, Gerd Klein, Michael Kneba, Dieter Köberle, Gabriela Kornek, Thomas Kühr, Ralf Küppers, Alois Lang, Florian Langer, Philipp le Coutre, Werner Linkesch, Andreas Lohri, Anja Lorch, Florian Lordick, Heinz Ludwig, Diana Lüftner, Andreas Mackensen, Robert Mader, Christine Mannhalter, Markus Manz, Christine Marosi, Anja Mehnert, Thomas Melchardt, Johannes Meran, Olaf Merkel, Brigitte Mlineritsch, Carsten Müller-Tidow, Andreas Neubauer, Dietger Niederwieser, Karin Oechsle, Aurelius Omlin, Oliver Ottmann, Friedrich Overkamp, Ingrid Pabinger, Jens Panse, Jakob Passweg, Christian Peschel, Bernhard-C. Pestalozzi, Christina Peters, Petro E. Petrides, Andreas Petzer, Holger Pfaff, Michael Pfeilstöcker, Robert Pirker, Ferdinand Ploner, Markus Raderer, Oliver Rick, Hanno Riess, Thomas Ruhstaller, Renate Schaberl-Moser, Brigitte Schallmoser, Werner Scheithauer, Richard F. Schlenk, Ingo Schmidt-Wolf, Clemens A. Schmitt, Norbert Schmitz, Jochen Schütte, Marco Siano, Michael Stahl, Reinhard Stauder, Jens Stäudle, Günther Steger, Alexander Stein, Michael Steurer, Stephan Stilgenbauer, Florian Strasser, Imke Strohscheer, Roger Stupp, Josef Thaler, Matthias Theobald, Christian Thiede, Eckhard Thiel, Nils Thoennissen, Peter Thuss-Patience, André Tichelli, Marlene Troch, Christof von Kalle, Herbert Watzke, Rudolf Weide, Ansgar Weltermann, Clemens-Martin Wendtner, Martin Wilhelm, Hansjochen Wilke, Daniel Winter, Mathias Witzens-Harig, Dominik Wolf, Bernhard J. Wörmann, Patrick Wuchter, Gerald Wulf, August Zabernigg, Robert Zeiser, Christoph Zielinski, Katja Zirlik Disclosure Statement The editors declare no conflict of interest.