Thomas Oellerich

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.

A transgenic mouse model demonstrating the oncogenic role of mutations in the polycomb-group gene EZH2 in lymphomagenesis

The histone methyltransferase EZH2 is frequently mutated in germinal center-derived diffuse large B-cell lymphoma and follicular lymphoma. To further characterize these EZH2 mutations in lymphomagenesis, we generated a mouse line where EZH2(Y641F) is expressed from a lymphocyte-specific promoter. Spleen cells isolated from the transgenic mice displayed a global increase in trimethylated H3K27, but the mice did not show an increased tendency to develop lymphoma. As EZH2 mutations often coincide with other mutations in lymphoma, we combined the expression of EZH2(Y641F) by crossing these transgenic mice with Eµ-Myc transgenic mice. We observed a dramatic acceleration of lymphoma development in this combination model of Myc and EZH2(Y641F). The lymphomas show histologic features of high-grade disease with a shift toward a more mature B-cell phenotype, increased cycling and gene expression, and epigenetic changes involving important pathways in B-cell regulation and function. Furthermore, they initiate disease in secondary recipients. In summary, EZH2(Y641F) can collaborate with Myc to accelerate lymphomagenesis demonstrating a cooperative role of EZH2 mutations in oncogenesis. This murine lymphoma model provides a new tool to study global changes in the epigenome caused by this frequent mutation and a promising model system for testing novel treatments.

Nck-mediated recruitment of BCAP to the BCR regulates the PI(3)K-Akt pathway in B cells

The adaptor Nck links receptor signaling to cytoskeleton regulation. Here we found that Nck also controlled the phosphatidylinositol-3-OH kinase (PI(3)K)–kinase Akt pathway by recruiting the adaptor BCAP after activation of B cells. Nck bound directly to the B cell antigen receptor (BCR) via the non–immunoreceptor tyrosine-based activation motif (ITAM) phosphorylated tyrosine residue at position 204 in the tail of the immunoglobulin-α component. Genetic ablation of Nck resulted in defective BCR signaling, which led to hampered survival and proliferation of B cells in vivo. Indeed, antibody responses in Nck-deficient mice were also considerably impaired. Thus, we demonstrate a previously unknown adaptor function for Nck in recruiting BCAP to sites of BCR signaling and thereby modulating the PI(3)K-Akt pathway in B cells.

Leukemic blasts program bone marrow adipocytes to generate a protumoral microenvironment

Despite currently available therapies, most patients diagnosed with acute myeloid leukemia (AML) die of their disease. Tumor-host interactions are critical for the survival and proliferation of cancer cells; accordingly, we hypothesize that specific targeting of the tumor microenvironment may constitute an alternative or additional strategy to conventional tumor-directed chemotherapy. Because adipocytes have been shown to promote breast and prostate cancer proliferation, and because the bone marrow adipose tissue accounts for up to 70% of bone marrow volume in adult humans, we examined the adipocyte-leukemia cell interactions to determine if they are essential for the growth and survival of AML. Using in vivo and in vitro models of AML, we show that bone marrow adipocytes from the tumor microenvironment support the survival and proliferation of malignant cells from patients with AML. We show that AML blasts alter metabolic processes in adipocytes to induce phosphorylation of hormone-sensitive lipase and consequently activate lipolysis, which then enables the transfer of fatty acids from adipocytes to AML blasts. In addition, we report that fatty acid binding protein-4 (FABP4) messenger RNA is upregulated in adipocytes and AML when in coculture. FABP4 inhibition using FABP4 short hairpin RNA knockdown or a small molecule inhibitor prevents AML proliferation on adipocytes. Moreover, knockdown of FABP4 increases survival in Hoxa9/Meis1-driven AML model. Finally, knockdown of carnitine palmitoyltransferase IA in an AML patient-derived xenograft model improves survival. Here, we report the first description of AML programming bone marrow adipocytes to generate a protumoral microenvironment.

Loss of the histone methyltransferase EZH2 induces resistance to multiple drugs in acute myeloid leukemia

In acute myeloid leukemia (AML), therapy resistance frequently occurs, leading to high mortality among patients. However, the mechanisms that render leukemic cells drug resistant remain largely undefined. Here, we identified loss of the histone methyltransferase EZH2 and subsequent reduction of histone H3K27 trimethylation as a novel pathway of acquired resistance to tyrosine kinase inhibitors (TKIs) and cytotoxic drugs in AML. Low EZH2 protein levels correlated with poor prognosis in AML patients. Suppression of EZH2 protein expression induced chemoresistance of AML cell lines and primary cells in vitro and in vivo. Low EZH2 levels resulted in derepression of HOX genes, and knockdown of HOXB7 and HOXA9 in the resistant cells was sufficient to improve sensitivity to TKIs and cytotoxic drugs. The endogenous loss of EZH2 expression in resistant cells and primary blasts from a subset of relapsed AML patients resulted from enhanced CDK1-dependent phosphorylation of EZH2 at Thr487. This interaction was stabilized by heat shock protein 90 (HSP90) and followed by proteasomal degradation of EZH2 in drug-resistant cells. Accordingly, inhibitors of HSP90, CDK1 and the proteasome prevented EZH2 degradation, decreased HOX gene expression and restored drug sensitivity. Finally, patients with reduced EZH2 levels at progression to standard therapy responded to the combination of bortezomib and cytarabine, concomitant with the re-establishment of EZH2 expression and blast clearance. These data suggest restoration of EZH2 protein as a viable approach to overcome treatment resistance in this AML patient population.

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.

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.

SLP-65 Phosphorylation Dynamics Reveals a Functional Basis for Signal Integration by Receptor-proximal Adaptor Proteins

Understanding intracellular signal transduction by cell surface receptors requires information about the precise order of relevant modifications on the early transducer elements. Here we introduce the B cell line DT40 and its genetically engineered variants as a model system to determine and functionally characterize post-translational protein modifications in general. This is accomplished by a customized strategy that combines mass spectrometric analyses of protein modifications with subsequent mutational studies. When applied to the B cell receptor (BCR)-proximal effector SLP-65, this approach uncovered a differential and highly dynamic engagement of numerous newly identified phospho-acceptor sites. Some of them serve as kinase substrates in resting cells and undergo rapid dephosphorylation upon BCR ligation. Stimulationinduced phosphorylation of SLP-65 can be early and transient, or early and sustained, or late. Functional elucidation of conspicuous phosphorylation at serine 170 in SLP-65 revealed a BCR-distal checkpoint for some but not all possible B cell responses. Our data show that SLP-65 phosphorylation acts upstream for signal initiation and also downstream during selective processing of the BCR signal. Such a phenomenon defines a receptor-specific signal integrator.