Hanibal Bohnenberger

The B-cell antigen receptor signals through a preformed transducer module of SLP65 and CIN85.

Spleen tyrosine kinase Syk and its substrate SLP65 (also called BLNK) are proximal signal transducer elements of the B‐cell antigen receptor (BCR). Yet, our understanding of signal initiation and processing is limited owing to the incomplete list of SLP65 interaction partners and our ignorance of their association kinetics. We have now determined and quantified the in vivo interactomes of SLP65 in resting and stimulated B cells by mass spectrometry. SLP65 orchestrated a complex signal network of about 30 proteins that was predominantly based on dynamic interactions. However, a stimulation‐independent and constant association of SLP65 with the Cbl‐interacting protein of 85 kDa (CIN85) was requisite for SLP65 phosphorylation and its inducible plasma membrane translocation. In the absence of a steady SLP65/CIN85 complex, BCR‐induced Ca2+ and NF‐κB responses were abrogated. Finally, live cell imaging and co‐immunoprecipitation experiments further confirmed that both SLP65 and CIN85 are key components of the BCR‐associated primary transducer module required for the onset and progression phases of BCR signal transduction.

SAMHD1 is a biomarker for cytarabine response and a therapeutic target in acute myeloid leukemia

The nucleoside analog cytarabine (Ara-C) is an essential component of primary and salvage chemotherapy regimens for acute myeloid leukemia (AML). After cellular uptake, Ara-C is converted into its therapeutically active triphosphate metabolite, Ara-CTP, which exerts antileukemic effects, primarily by inhibiting DNA synthesis in proliferating cells. Currently, a substantial fraction of patients with AML fail to respond effectively to Ara-C therapy, and reliable biomarkers for predicting the therapeutic response to Ara-C are lacking. SAMHD1 is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase that cleaves physiological dNTPs into deoxyribonucleosides and inorganic triphosphate. Although it has been postulated that SAMHD1 sensitizes cancer cells to nucleoside-analog derivatives through the depletion of competing dNTPs, we show here that SAMHD1 reduces Ara-C cytotoxicity in AML cells. Mechanistically, dGTP-activated SAMHD1 hydrolyzes Ara-CTP, which results in a drastic reduction of Ara-CTP in leukemic cells. Loss of SAMHD1 activity—through genetic depletion, mutational inactivation of its triphosphohydrolase activity or proteasomal degradation using specialized, virus-like particles—potentiates the cytotoxicity of Ara-C in AML cells. In mouse models of retroviral AML transplantation, as well as in retrospective analyses of adult patients with AML, the response to Ara-C-containing therapy was inversely correlated with SAMHD1 expression. These results identify SAMHD1 as a potential biomarker for the stratification of patients with AML who might best respond to Ara-C-based therapy and as a target for treating Ara-C-refractory AML.

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.

STAT5-regulated microRNA-193b controls haematopoietic stem and progenitor cell expansion by modulating cytokine receptor signalling.

Haematopoietic stem cells (HSCs) require the right composition of microRNAs (miR) for proper life-long balanced blood regeneration. Here we show a regulatory circuit that prevents excessive HSC self-renewal by upregulation of miR-193b upon self-renewal promoting thrombopoietin (TPO)-MPL-STAT5 signalling. In turn, miR-193b restricts cytokine signalling, by targeting the receptor tyrosine kinase c-KIT. We generated a miR-193b knockout mouse model to unravel the physiological function of miR-193b in haematopoiesis. MiR-193b−/− mice show a selective gradual enrichment of functional HSCs, which are fully competent in multilineage blood reconstitution upon transplantation. The absence of miR-193b causes an accelerated expansion of HSCs, without altering cell cycle or survival, but by decelerating differentiation. Conversely, ectopic miR-193b expression restricts long-term repopulating HSC expansion and blood reconstitution. MiR-193b-deficient haematopoietic stem and progenitor cells exhibit increased basal and cytokine-induced STAT5 and AKT signalling. This STAT5-induced microRNA provides a negative feedback for excessive signalling to restrict uncontrolled HSC expansion.

Complex phosphorylation dynamics control the composition of the Syk interactome in B cells.

Spleen tyrosine kinase Syk provides critical transducer functions for a number of immune cell receptors and has been implicated in the generation of several forms of leukemias. Catalytic activity and the ability of Syk to interact with other signaling elements depend on the phosphorylation status of Syk. We have now identified and quantified the full spectrum of phosphoacceptor sites in human Syk as well as the interactome of Syk in resting and activated B cells by high‐resolution mass spectrometry. While the majority of inducible phosphorylations occurred on tyrosine residues, one of the most frequently detected phosphosites encompassed serine 297 located within the linker insert distinguishing the long and short isoforms of Syk. Full‐length Syk can associate with more than 25 distinct ligands including the 14‐3‐3γ adaptor protein, which binds directly to phosphoserine 297. The latter complex attenuates inducible plasma membrane recruitment of Syk, thereby limiting antigen receptor‐proximal signaling pathways. Collectively, the established ligand library provides a basis to understand the complexity of the Syk signaling network.

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.

The Dok-3/Grb2 Protein Signal Module Attenuates Lyn Kinase-dependent Activation of Syk Kinase in B Cell Antigen Receptor Microclusters

Background: The signal module comprising Dok-3 and Grb2 controls differential BCR signal intensity. Results: Dok-3/Grb2 translocate to BCR microsignalosomes and inhibit Lyn-dependent activation of the BCR transducer kinase Syk. Conclusion: Dok-3/Grb2 change the balance of activatory and inhibitory Lyn functions toward BCR signal inhibition. Significance: Learning how adapter proteins translocate to and change signal processes in BCR microsignalosomes is important to understand the regulation of antigen-induced B cell activation. Recruitment of the growth factor receptor-bound protein 2 (Grb2) by the plasma membrane-associated adapter protein downstream of kinase 3 (Dok-3) attenuates signals transduced by the B cell antigen receptor (BCR). Here we describe molecular details of Dok-3/Grb2 signal integration and function, showing that the Lyn-dependent activation of the BCR transducer kinase Syk is attenuated by Dok-3/Grb2 in a site-specific manner. This process is associated with the SH3 domain-dependent translocation of Dok-3/Grb2 complexes into BCR microsignalosomes and augmented phosphorylation of the inhibitory Lyn target SH2 domain-containing inositol 5′ phosphatase. Hence, our findings imply that Dok-3/Grb2 modulates the balance between activatory and inhibitory Lyn functions with the aim to adjust BCR signaling efficiency.

Aneurysm of the Pulmonary Vein: An Unusual Cause of Stroke

This clinical report deals with a giant true pulmonary venous aneurysm, which was partially thrombosed. The overall incidence of pulmonary venous aneurysms is unknown, and they are reported only occasionally. We present the case of a previously healthy man with acute onset of ischemic cerebral stroke. The cause was a thrombus in a huge aneurysm of the left superior pulmonary vein. The patient subsequently underwent uncomplicated therapy for stroke, including thrombolysis followed by excision of the giant pulmonary venous aneurysm. As curative therapy we recommend complete resection of this rare entity.