Search

Burkitt lymphoma (BL) is an aggressive lymphoma type that is currently treated by intensive chemoimmunotherapy. Despite the favorable clinical outcome for most patients with BL, chemotherapy-related toxicity and disease relapse remain major clinical challenges, emphasizing the need for innovative therapies. Using genome-scale CRISPR-Cas9 screens, we identified B-cell receptor (BCR) signaling, specific transcriptional regulators, and one-carbon metabolism as vulnerabilities in BL. We focused on serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism. Inhibition of SHMT2 by either knockdown or pharmacological compounds induced anti-BL effects in vitro and in vivo. Mechanistically, SHMT2 inhibition led to a significant reduction of intracellular glycine and formate levels, which inhibited the mTOR pathway and thereby triggered autophagic degradation of the oncogenic transcription factor TCF3. Consequently, this led to a collapse of tonic BCR signaling, which is controlled by TCF3 and is essential for BL cell survival. In terms of clinical translation, we also identified drugs such as methotrexate that synergized with SHMT inhibitors. Overall, our study has uncovered the dependency landscape in BL, identified and validated SHMT2 as a drug target, and revealed a mechanistic link between SHMT2 and the transcriptional master regulator TCF3, opening up new perspectives for innovative therapies.

Despite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein–protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.

Epstein-Barr virus (EBV) infects human B cells and reprograms them to allow virus replication and persistence. One key viral factor in this process is latent membrane protein 2A (LMP2A), which has been described as a B cell receptor (BCR) mimic promoting malignant transformation. However, how LMP2A signaling contributes to tumorigenesis remains elusive. By comparing LMP2A and BCR signaling in primary human B cells using phosphoproteomics and transcriptome profiling, we identified molecular mechanisms through which LMP2A affects B cell biology. Consistent with the literature, we found that LMP2A mimics a subset of BCR signaling events, including tyrosine phosphorylation of the kinase SYK, the calcium initiation complex consisting of BLNK, BTK, and PLCγ2, and its downstream transcription factor NFAT. However, the majority of LMP2A-induced signaling events markedly differed from those induced by BCR stimulation. These included differential phosphorylation of kinases, phosphatases, adaptor proteins, transcription factors such as nuclear factor κB (NF-κB) and TCF3, as well as widespread changes in the transcriptional output of LMP2A-expressing B cells. LMP2A affected apoptosis and cell-cycle checkpoints by dysregulating the expression of apoptosis regulators such as BCl-xL and the tumor suppressor retinoblastoma-associated protein 1 (RB1). LMP2A cooperated with MYC and mutant cyclin D3, two oncogenic drivers of Burkitt lymphoma, to promote proliferation and survival of primary human B cells by counteracting MYC-induced apoptosis and by inhibiting RB1 function, thereby promoting cell-cycle progression. Our results indicate that LMP2A is not a pure BCR mimic but rather rewires intracellular signaling in EBV-infected B cells that optimizes cell survival and proliferation, setting the stage for oncogenic transformation.

Abstract Multiple myeloma patients are often treated with immunomodulatory drugs, proteasome inhibitors, or monoclonal antibodies until disease progression. Continuous therapy in combination with the underlying disease frequently results in severe humoral and cellular immunodeficiency, which often manifests in recurrent infections. Here, we report on the clinical management and immunological data of three multiple-myeloma patients diagnosed with COVID-19. Despite severe hypogammaglobulinemia, deteriorated T cell counts, and neutropenia, the patients were able to combat COVID-19 by balanced response of innate immunity, strong CD8+ and CD4+ T cell activation and differentiation, development of specific T-cell memory subsets, and development of anti-SARS-CoV-2 type IgM and IgG antibodies with virus-neutralizing capacities. Even 12 months after re-introduction of lenalidomide maintenance therapy, antibody levels and virus-neutralizing antibody titers remained detectable, indicating persisting immunity against SARS-CoV-2. We conclude that in MM patients who tested positive for SARS-CoV-2 and were receiving active MM treatment, immune response assessment could be a useful tool to help guide decision-making regarding the continuation of anti-tumor therapy and supportive therapy. Key messages Immunosuppression due to multiple myeloma might not be the crucial factor that is affecting the course of COVID-19. In this case, despite pre-existing severe deficits in CD4+ T-cell counts and IgA und IgM deficiency, we noticed a robust humoral and cellular immune response against SARS-CoV-2. Evaluation of immune response and antibody titers in MM patients that were tested positive for SARS-CoV-2 and are on active MM treatment should be performed on a larger scale; the findings might affect further treatment recommendations for COVID-19, MM treatment re-introduction, and isolation measures.

Cancer-related treatment is associated with development of heart failure and poor outcome in cancer-survivors. T1 and T2 mapping by cardiovascular magnetic resonance (CMR) may detect myocardial injury due to cancer-related treatment.Patients receiving cancer-related treatment regimes underwent screening of cardiac involvement with CMR, either within 3 months (early Tx) or12 months (late Tx) post-treatment. T1 and T2 mapping, cardiac function, strain, ischaemia-testing, scar-imaging and serological cardiac biomarkers were obtained.Compared to age/gender matched controls (n = 57), patients (n = 115, age (yrs): median(IQR) 48(28-60), females, n = 60(52%) had reduced left ventricular ejection fraction (LV-EF) and strain, and higher native T1 and T2. The early Tx group (n = 52) had significantly higher native T1, T2 and troponin levels compared to the late Tx group, indicating myocardial inflammation and oedema (p 0.01). On the contrary, late Tx patients showed raised native T1, increased LV-end-systolic volumes, reduced LV-EF and deformation, and elevated NT-proBNP, suggesting myocardial fibrosis and remodelling (p 0.05). Prospective validation of these results in an independent cohort of patients with similar treatment regimens (n = 25) and longitudinal assessments revealed high concordance of CMR imaging signatures of early and late cardiac involvement.Native T1 and T2 mapping can be valuable in detecting and monitoring of cardiac involvement with cancer-related treatment, providing distinct biosignatures of early inflammatory involvement (raised native T1 and T2) and interstitial fibrosis and remodelling (raised native T1 but not T2), respectively. Our findings may provide an algorithm allowing to identify susceptible myocardium to potentially guide cardio-protective treatment measures.

Recent studies implicated that long non-coding RNAs (lncRNAs) may play a role in the progression and development of acute lymphoblastic leukemia, however, this role is not yet clear. In order to unravel the role of lncRNAs associated with B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL) subtypes, we performed transcriptome sequencing and DNA methylation array across 82 BCP-ALL samples from three molecular subtypes (DUX4, Ph-like, and Near Haploid or High Hyperdiploidy). Unsupervised clustering of BCP-ALL samples on the basis of their lncRNAs on transcriptome and DNA methylation profiles revealed robust clusters separating three molecular subtypes. Using extensive computational analysis, we developed a comprehensive catalog of 1235 aberrantly dysregulated BCP-ALL subtype-specific lncRNAs with altered expression and methylation patterns from three subtypes of BCP-ALL. By analyzing the co-expression of subtype-specific lncRNAs and protein-coding genes, we inferred key molecular processes in BCP-ALL subtypes. A strong correlation was identified between the DUX4 specific lncRNAs and activation of TGF-β and Hippo signaling pathways. Similarly, Ph-like specific lncRNAs were correlated with genes involved in activation of PI3K-AKT, mTOR, and JAK-STAT signaling pathways. Interestingly, the relapse-specific differentially expressed lncRNAs correlated with the activation of metabolic and signaling pathways. Finally, we showed a set of epigenetically altered lncRNAs facilitating the expression of tumor genes located at theircislocation. Overall, our study provides a comprehensive set of novel subtype and relapse-specific lncRNAs in BCP-ALL. Our findings suggest a wide range of molecular pathways are associated with lncRNAs in BCP-ALL subtypes and provide a foundation for functional investigations that could lead to new therapeutic approaches.Author SummaryAcute lymphoblastic leukemia is a heterogeneous blood cancer, with multiple molecular subtypes, and with high relapse rate. We are far from the complete understanding of the rationale behind these subtypes and high relapse rate. Long non-coding (lncRNAs) has emerged as a novel class of RNA due to its diverse mechanism in cancer development and progression. LncRNAs does not code for proteins and represent around 70% of human transcripts. Recently, there are a number of studies used lncRNAs expression profile in the classification of various cancers subtypes and displayed their correlation with genomic, epigenetic, pathological and clinical features in diverse cancers. Therefore, lncRNAs can account for heterogeneity and has independent prognostic value in various cancer subtypes. However, lncRNAs defining the molecular subtypes of BCP-ALL are not portrayed yet. Here, we describe a set of relapse and subtype-specific lncRNAs from three major BCP-ALL subtypes and define their potential functions and epigenetic regulation. Our data uncover the diverse mechanism of action of lncRNAs in BCP-ALL subtypes defining how lncRNAs are involved in the pathogenesis of disease and the relevance in the stratification of BCP-ALL subtypes.

Somatic mutations in DNA Methyltransferase 3A (DNMT3A) are among the most frequent alterations in clonal hematopoiesis (CH) and Acute Myeloid Leukemia (AML), with a hotspot in exon 23 at arginine 882 (DNMT3A-R882). Here we demonstrate that DNMT3A-R882H-dependent CH- and AML cells are specifically susceptible to the hypomethylating agent azacytidine (AZA). Addition of AZA to chemotherapy prolonged AML survival solely in patients with DNMT3A-R882 mutation, suggesting its potential as a novel predictive marker for AZA response. AML and CH mouse models confirmed AZA susceptibility specifically in DNMT3A-R882H-expressing cells. Hematopoietic stem and progenitor cells expressing DNMT3A-R882H exhibited cell-autonomous viral mimicry response as a result of focal DNA hypomethylation at retrotransposon sequences. Administration of AZA boosted hypomethylation of retrotransposons specifically in DNMT3A-R882H expressing cells and maintained elevated levels of canonical interferon-stimulated genes (ISGs), thus leading to suppressed protein translation and increased apoptosis.

Mycobacterium abscessus is a highly antibiotic-resistant opportunistic pathogen causing clinically challenging infections in patients with preexisting lung diseases or under immunosuppression. Hence, reliable antibiotic susceptibility data are required for effective treatment. Aims of this study were to investigate (i) the congruence of genotypic and phenotypic antimicrobial susceptibility testing, (ii) the relationship between resistance profile and clinical course, and (iii) the phylogenetic relations of M. abscessus in a German patient cohort. A total of 39 isolates from 29 patients infected or colonized with M. abscessus underwent genotypic and phenotypic drug susceptibility testing. Clinical data were correlated with susceptibility data. Phylogenetic analysis was performed by means of whole-genome sequencing (WGS) and single-nucleotide polymorphism (SNP) analysis. Macrolide resistance was mainly mediated by functional Erm(41) methyltransferases (T28 sequevars) in M. abscessus subsp. abscessus (n = 25) and M. abscessus subsp. bolletii (n = 2). It was significantly associated with impaired culture conversion (P = 0.02). According to the core SNP phylogeny, we identified three clusters of closely related isolates with SNP distances below 25. Representatives of all circulating global clones (Absc. 1, Absc. 2, and Mass. 1) were identified in our cohort. However, we could not determine evidence for in-hospital interhuman transmission from clinical data. In our patient cohort, we identified three M. abscessus clusters with closely related isolates and representatives of the previously described international clusters but no human-to-human in-hospital transmission. Macrolide and aminoglycoside susceptibility data are critical for therapeutic decision-making in M. abscessus infections.

Acute myeloid leukemia (AML) is a heterogeneous group of diseases caused by oncogenic transformation of hematopoietic stem and progenitor cells. Mutations in the gene encoding the tyrosine kinase FLT3 leading to internal tandem duplications (ITDs) of sequence represent one of the most frequent

BACKGROUND Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment option for patients with acute myeloid leukemia (AML). During transplantation, patients undergo a period of severe neutropenia, which puts them at high risk for infectious complications. However, the impact of patient colonization with multidrug-resistant organisms (MDRO) on overall survival remains unclear. METHODS In this retrospective, single-center study, the authors analyzed data from 264 patients with AML who underwent a first allo-HSCT between January 2006 and March 2016 at their institution. Primary endpoints were overall survival and nonrelapse-related mortality. RESULTS One hundred forty-two of 264 patients (53.8%) were colonized by at least 1 MDRO, mainly with vancomycin-resistant Enterococcus faecalis/faecium (n = 122). The characteristics of colonized patients did not differ from those of MDRO-negative patients with respect to median age (53.5 vs 53 years), cytogenetic risk according to European LeukemiaNet criteria, remission status before allo-HSCT (first or second complete remission: 55.7% vs 60.7%, respectively; active disease: 44.4% vs 39.3%, respectively), donor type, or hematopoietic cell transplantation-comorbidity index (HCT-CI). Compared with noncolonized patients, MDRO-positive patients had an inferior probability of survival at 5 years (43.3% vs 65.5%; P = .002), primarily because of a higher cumulative incidence of nonrelapse-related mortality (33.9% vs 9.4%; P < .001). Death caused by infections occurred in 15.5% of colonized patients versus 4.9% of noncolonized patients. There was no difference in the cumulative incidence of relapse in MDRO-positive versus MDRO-negative patients (33.8% vs 42.1%, respectively; P = .798). CONCLUSIONS The current data emphasize the importance of regular MDRO screenings and prompt further investigations into the impact of colonization with MDRO on the immune system after allo-HSCT. Cancer 2018;124:286-96. © 2017 American Cancer Society.

Donor cell leukemia: evidence for multiple preleukemic clones and parallel long term clonal evolution in donor and recipient

Background Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide. Growing evidence indicates that tumor-initiating cells (TICs) are responsible for tumor growth and progression. Conventional chemotherapeutics do not sufficiently eliminate TICs, leading to tumor relapse. We aimed to gain insight into TIC biology by comparing the transcriptome of primary TIC cultures and their normal stem cell counterparts to uncover expression differences. Methods We established colonosphere cultures derived from the resection of paired specimens of primary tumor and normal mucosa in patients with CRC. These colonospheres, enriched for TICs, were used for differential transcriptome analyses to detect new targets for a TIC-directed therapy. Effects of target inhibition on CRC cells were studied in vitro and in vivo. Results Pathway analysis of the regulated genes showed enrichment of genes central to PI3K/AKT and Wnt-signaling. We identified CD133 as a marker for a more aggressive CRC subpopulation enriched with TICs in SW480 CRC cells in an in vivo cancer model. Treatment of CRC cells with the selective AKT inhibitor MK-2206 caused a decrease in cell proliferation, particularly in the TIC fraction, resulting in a significant reduction of the stemness capacity to form colonospheres in vitro and to initiate tumor formation in vivo. Consequently, MK-2206 treatment of mice with established xenograft tumors exhibited a significant deceleration of tumor progression. Primary patient-derived tumorsphere growth was significantly inhibited by MK-2206. Conclusion This study reveals that AKT signaling is critical for TIC proliferation and can be efficiently targeted by MK-2206 representing a preclinical therapeutic strategy to repress colorectal TICs. Electronic supplementary material The online version of this article (doi:10.1245/s10434-016-5218-z) contains supplementary material, which is available to authorized users.

LSD1 has emerged as a promising epigenetic target in the treatment of acute myeloid leukemia (AML). We used two murine AML models based on retroviral overexpression of Hoxa9/Meis1 (H9M) or MN1 to study LSD1 loss of function in AML. The conditional knockout of Lsd1 resulted in differentiation with both granulocytic and monocytic features and increased ATRA sensitivity and extended the survival of mice with H9M-driven AML. The conditional knockout led to an increased expression of multiple genes regulated by the important myeloid transcription factors GFI1 and PU.1. These include the transcription factors GFI1B and IRF8. We also compared the effect of different irreversible and reversible inhibitors of LSD1 in AML and could show that only tranylcypromine derivatives were capable of inducing a differentiation response. We employed a conditional knock-in model of inactive, mutant LSD1 to study the effect of only interfering with LSD1 enzymatic activity. While this was sufficient to initiate differentiation, it did not result in a survival benefit in mice. Hence, we believe that targeting both enzymatic and scaffolding functions of LSD1 is required to efficiently treat AML. This finding as well as the identified biomarkers may be relevant for the treatment of AML patients with LSD1 inhibitors.

Summary FLT3 and c-KIT are crucial regulators of hematopoietic stem and progenitor cells. We investigated the role of STS1 and STS2 on FLT3 and c-KIT phosphorylation, activity, and function in normal and stress-induced hematopoiesis. STS1/STS2-deficient mice show a profound expansion of multipotent progenitor and lymphoid primed multipotent progenitor cells with elevated colony-forming capacity. Although long-term hematopoietic stem cells are not increased in numbers, lack of STS1 and STS2 significantly promotes long-term repopulation activity, demonstrating a pivotal role of STS1/STS2 in regulating hematopoietic stem and progenitor cell fitness. Biochemical analysis identified STS1/STS2 as direct phosphatases of FLT3 and c-KIT. Loss of STS1/STS2 induces hyperphosphorylation of FLT3, enhances AKT signaling, and confers a strong proliferative advantage. Therefore, our study reveals that STS1 and STS2 may serve as novel pharmaceutical targets to improve hematopoietic recovery after bone marrow transplantation., Graphical Abstract, Highlights • Hematopoietic stem cells lacking STS1/STS2 display significantly enhanced fitness • STS1 and STS2 are phosphatases of FLT3 and c-KIT • Lack of STS1/STS2 activates FLT3 and downstream signaling, Brandts and colleagues report a novel mechanism of hematopoietic stem and progenitor cell regulation by phosphatases. Mice lacking STS1 and STS2 show a significant increase in hematopoietic stem cell fitness and long-term repopulation capacity as well as enhanced progenitor cell proliferation. The authors identify STS1/STS2 as direct phosphatases of FLT3, regulating downstream signaling and cell proliferation.

Activating mutations of FMS-like tyrosine kinase 3 (FLT3), notably internal tandem duplications (ITDs), are associated with a grave prognosis in acute myeloid leukemia (AML). Transforming FLT3ITD signal transduction causes formation of reactive oxygen species (ROS) and inactivation of the protein-tyrosine phosphatase (PTP) DEP-1/PTPRJ, a negative regulator of FLT3 signaling. Here we addressed the underlying mechanisms and biological consequences. NADPH oxidase 4 (NOX4) messenger RNA and protein expression was found to be elevated in FLT3ITD-positive cells and to depend on FLT3ITD signaling and STAT5-mediated activation of the NOX4 promoter. NOX4 knockdown reduced ROS levels, restored DEP-1 PTP activity and attenuated FLT3ITD-driven transformation. Moreover, Nox4 knockout (Nox4(-/-)) murine hematopoietic progenitor cells were refractory to FLT3ITD-mediated transformation in vitro. Development of a myeloproliferative-like disease (MPD) caused by FLT3ITD-transformed 32D cells in C3H/HeJ mice, and of a leukemia-like disease in mice transplanted with MLL-AF9/ FLT3ITD-transformed murine hematopoietic stem cells were strongly attenuated by NOX4 downregulation. NOX4-targeting compounds were found to counteract proliferation of FLT3ITD-positive AML blasts and MPD development in mice. These findings reveal a previously unrecognized mechanism of oncoprotein-driven PTP oxidation, and suggest that interference with FLT3ITD-STAT5-NOX4-mediated overproduction of ROS and PTP inactivation may have therapeutic potential in a subset of AML.

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.

Acute myeloid leukemia in the elderly is characterized by a distinct genetic and epigenetic landscape

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(2,3). SAMHD1 is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase that cleaves physiological dNTPs into deoxyribonucleosides and inorganic triphosphate(4,5). Although it has been postulated that SAMHD1 sensitizes cancer cells to nucleoside-analog derivatives through the depletion of competing dNTPs6, 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(7,8) 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.

Leukemia, 31 (3), ISSN:1476-5551, ISSN:0887-6924

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.

Acute myeloid leukemia (AML) is one of the most common and life-threatening leukemias. A highly diverse and flexible metabolism contributes to the aggressiveness of the disease that is still difficult to treat. By using different sources of nutrients for energy and biomass supply, AML cells gain metabolic plasticity and rapidly outcompete normal hematopoietic cells. This review aims to decipher the diverse metabolic strategies and the underlying oncogenic and environmental changes that sustain continuous growth, mediate redox homeostasis and induce drug resistance in AML. We revisit Warburg’s hypothesis and illustrate the role of glucose as a provider of cellular building blocks rather than as a supplier of the tricarboxylic acid (TCA) cycle for energy production. We discuss how the diversity of fuels for the TCA cycle, including glutamine and fatty acids, contributes to the metabolic plasticity of the disease and highlight the roles of amino acids and lipids in AML metabolism. Furthermore, we point out the potential of the different metabolic effectors to be used as novel therapeutic targets.

Ag-mediated B cell stimulation relies on phospholipase Cγ2 (PLCγ2) for Ca2+ mobilization. Enzymatic activity of PLCγ2 is triggered upon Src homology 2 domain–mediated binding to the tyrosine-phosphorylated adaptor SLP65. However, SLP65 phosphorylation outlasts the elevation of cytosolic Ca2+ concentration suggesting additional levels of PLCγ2 regulation. We show in this article that the functionality of the PLCγ2/SLP65 complex is controlled by the weakly characterized C2 domain of PLCγ2. Usually C2 domains bind membrane lipids, but that of PLCγ2 docks in a Ca2+-regulated manner to a distinct phosphotyrosine of SLP65. Hence, early Ca2+ fluxing provides feed-forward signal amplification by promoting anchoring of the PLCγ2 C2 domain to phospho-SLP65. As the cellular Ca2+ resources become exhausted, the concomitant decline of Ca2+ dampens the C2-phosphotyrosine interaction so that PLCγ2 activation terminates despite sustained SLP65 phosphorylation.

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.

Behrens et al. establish the interplay of activated FLT3 receptor and the phosphorylated RUNX1 transcription factor in uncoupling proliferation and differentiation signals in acute leukemia. These findings demonstrate that RUNX1 is a viable therapeutic target in FLT3-mutated AML., Acute myeloid leukemia (AML) is induced by the cooperative action of deregulated genes that perturb self-renewal, proliferation, and differentiation. Internal tandem duplications (ITDs) in the FLT3 receptor tyrosine kinase are common mutations in AML, confer poor prognosis, and stimulate myeloproliferation. AML patient samples with FLT3-ITD express high levels of RUNX1, a transcription factor with known tumor-suppressor function. In this study, to understand this paradox, we investigated the impact of RUNX1 and FLT3-ITD coexpression. FLT3-ITD directly impacts on RUNX1 activity, whereby up-regulated and phosphorylated RUNX1 cooperates with FLT3-ITD to induce AML. Inactivating RUNX1 in tumors releases the differentiation block and down-regulates genes controlling ribosome biogenesis. We identified Hhex as a direct target of RUNX1 and FLT3-ITD stimulation and confirmed high HHEX expression in FLT3-ITD AMLs. HHEX could replace RUNX1 in cooperating with FLT3-ITD to induce AML. These results establish and elucidate the unanticipated oncogenic function of RUNX1 in AML. We predict that blocking RUNX1 activity will greatly enhance current therapeutic approaches using FLT3 inhibitors.

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., Graphical Abstract, Highlights • Meis1 regulates expression and activation of Syk in Hoxa9-driven leukemia • Syk upregulation in Hoxa9/Meis1-driven leukemia is mediated by miR-146a • Increased Syk activity induces a Meis1 transcriptional program • In vivo, Meis1 sensitizes Hoxa9-driven leukemia to Syk inhibition, Mohr et al. identify a Meis1-dependent regulatory loop involving PU.1, miR-146a, and Syk, resulting in the addiction to Syk activity in Hoxa9/Meis1-transformed myeloid progenitor cells. Syk inhibition disrupts the regulatory loop and prolongs survival of mice with Hoxa9/Meis1-driven acute myeloid leukemia.

Burkitt's 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.

A complex aberrant karyotype consisting of multiple unrelated cytogenetic abnormalities is associated with poor prognosis in patients with acute myeloid leukemia (AML). The European Leukemia Net classification and the UK Medical Research Council recommendation provide prognostic categories that differ in the definition of unbalanced aberrations as well as the number of single aberrations. The aim of this study on 3526 AML patients was to redefine and validate a cutoff for karyotype complexity in AML with regard to adverse prognosis. Our study demonstrated that (1) patients with a pure hyperdiploid karyotype have an adverse risk irrespective of the number of chromosomal gains, (2) patients with translocation t(9;11)(p21∼22;q23) have an intermediate risk independent of the number of additional aberrations, (3) patients with ⩾4 abnormalities have an adverse risk per se and (4) patients with three aberrations in the absence of abnormalities of strong influence (hyperdiploid karyotype, t(9;11)(p21∼22;q23), CBF-AML, unique adverse-risk aberrations) have borderline intermediate/adverse risk with a reduced overall survival compared with patients with a normal karyotype.

The karyotype is so far the most important prognostic parameter in acute myeloid leukemia (AML). Molecular mutations have been analyzed to subdivide AML with normal karyotype into prognostic subsets. The aim of this study was to develop a prognostic model for the entire AML cohort solely based on molecular markers. One thousand patients with cytogenetic data were investigated for the following molecular alterations: PML-RARA, RUNX1-RUNX1T1, CBFB-MYH11, FLT3-ITD, and MLL-PTD, as well as mutations in NPM1, CEPBA, RUNX1, ASXL1, and TP53. Clinical data were available in 841 patients. Based on Cox regression and Kaplan-Meier analyses, 5 distinct prognostic subgroups were identified: (1) very favorable: PML-RARA rearrangement (n = 29) or CEPBA double mutations (n = 42; overall survival [OS] at 3 years: 82.9%); (2) favorable: RUNX1-RUNX1T1 (n = 35), CBFB-MYH11 (n = 31), or NPM1 mutation without FLT3-ITD (n = 186; OS at 3 years: 62.6%); (3) intermediate: none of the mutations leading to assignment into groups 1, 2, 4, or 5 (n = 235; OS at 3 years: 44.2%); (4) unfavorable: MLL-PTD and/or RUNX1 mutation and/or ASXL1 mutation (n = 203; OS at 3 years: 21.9%); and (5) very unfavorable: TP53 mutation (n = 80; OS at 3 years: 0%; P < .001). This comprehensive molecular characterization provides a more powerful model for prognostication than cytogenetics.

Acute myeloid leukemia (AML) is a highly malignant disease that is not curable in the majority of patients. Numerous non-random genetic abnormalities are known, among which several translocations such as PLZF/RARα or AML1/ETO are known to aberrantly recruit histone deacetylases. Deacetylase inhibitors (DACi) are promising drugs leading to growth inhibition, cell cycle arrest, premature senescence and apoptosis in malignant cells. It is believed that DACi may have clinical efficacy by eradicating the most primitive population of leukemic stem and progenitor cells, possibly by interfering with self-renewal. The aim of the study was to investigate the effects of DACi on leukemic stem and progenitor cells using murine transduction-transplantation models of hematopoietic cells harboring the leukemia-associated fusion proteins (LAFP) PLZF/RARα or a truncated AML1/ETO protein (AML1/ETO exon 9). We show that the self-renewal and short-term repopulation capacity of AML1/ETO- or PLZF/RARα-expressing Sca1+/lin- stem and progenitor cells are profoundly inhibited by clinically applicable concentrations of the DACi dacinostat and vorinostat. To further investigate the mechanisms underlying these effects, we examined the impact of DACi on the transcription factor c-MYC and the Polycomb group protein BMI1, which are induced by LAFP and involved in leukemic transformation. In AML1/ETO or PLZF/RARα-positive 32D cells, DACi-mediated antiproliferative effects were associated with downregulation of BMI1 and c-MYC protein levels. Similar effects were demonstrated in primary samples of cytogenetically defined high-risk AML patients. In conclusion, DACi may be effective as maintenance therapy by negatively interfering with signaling pathways that control survival and proliferation of leukemic stem and progenitor cells.

With the use of ChIP on microarray assays in primary leukemia samples, we report that acute myeloid leukemia (AML) blasts exhibit significant alterations in histone H3 acetylation (H3Ac) levels at > 1000 genomic loci compared with CD34+ progenitor cells. Importantly, core promoter regions tended to have lower H3Ac levels in AML compared with progenitor cells, which suggested that a large number of genes are epigenetically silenced in AML. Intriguingly, we identified peroxiredoxin 2 (PRDX2) as a novel potential tumor suppressor gene in AML. H3Ac was decreased at the PRDX2 gene promoter in AML, which correlated with low mRNA and protein expression. We also observed DNA hypermethylation at the PRDX2 promoter in AML. Low protein expression of the antioxidant PRDX2 gene was clinically associated with poor prognosis in patients with AML. Functionally, PRDX2 acted as inhibitor of myeloid cell growth by reducing levels of reactive oxygen species (ROS) generated in response to cytokines. Forced PRDX2 expression inhibited c-Myc–induced leukemogenesis in vivo on BM transplantation in mice. Taken together, epigenome-wide analyses of H3Ac in AML led to the identification of PRDX2 as an epigenetically silenced growth suppressor, suggesting a possible role of ROS in the malignant phenotype in AML.

Seroconversion rates following influenza vaccination in patients with hematologic malignancies after hematopoietic stem cell transplantation (HSCT) are known to be lower compared to healthy adults. The aim of our diagnostic study was to determine the rate of seroconversion after 1 or 2 doses of a novel split virion, inactivated, AS03-adjuvanted pandemic H1N1 influenza vaccine (A/California/7/2009) in HSCT recipients (ClinicalTrials.gov Identifier: NCT01017172). Blood samples were taken before and 21 days after a first dose and 21 days after a second dose of the vaccine. Antibody (AB) titers were determined by hemagglutination inhibition assay. Seroconversion was defined by either an AB titer of ≤ 1:10 before and ≥ 1:40 after or ≥ 1:10 before and ≥ 4-fold increase in AB titer 21 days after vaccination. Seventeen patients (14 allogeneic, 3 autologous HSCT) received 1 dose and 11 of these patients 2 doses of the vaccine. The rate of seroconversion was 41.2% (95% confidence interval [CI] 18.4-67.1) after the first and 81.8% (95% CI 48.2-97.7) after the second dose. Patients who failed to seroconvert after 1 dose of the vaccine were more likely to receive any immunosuppressive agent (P = .003), but time elapsed after or type of HSCT, age, sex, or chronic graft-versus-host disease was not different when compared to patients with seroconversion. In patients with hematologic malignancies after HSCT the rate of seroconversion after a first dose of an adjuvanted H1N1 influenza A vaccine was poor, but increased after a second dose.

The cell cycle is driven by the kinase activity of cyclin·cyclin-dependent kinase (CDK) complexes, which is negatively regulated by CDK inhibitor proteins. Recently, we identified INCA1 as an interaction partner and a substrate of cyclin A1 in complex with CDK2. On a functional level, we identified a novel cyclin-binding site in the INCA1 protein. INCA1 inhibited CDK2 activity and cell proliferation. The inhibitory effects depended on the cyclin-interacting domain. Mitogenic and oncogenic signals suppressed INCA1 expression, whereas it was induced by cell cycle arrest. We established a deletional mouse model that showed increased CDK2 activity in spleen with altered spleen architecture in Inca1(-/-) mice. Inca1(-/-) embryonic fibroblasts showed an increase in the fraction of S-phase cells. Furthermore, blasts from acute lymphoid leukemia and acute myeloid leukemia patients expressed significantly reduced INCA1 levels highlighting its relevance for growth control in vivo. Taken together, this study identifies a novel CDK inhibitor with reduced expression in acute myeloid and lymphoid leukemia. The molecular events that control the cell cycle occur in a sequential process to ensure a tight regulation, which is important for the survival of a cell and includes the detection and repair of genetic damage and the prevention of uncontrolled cell division.

Epigenetic changes play a crucial role in leukemogenesis. HDACs are frequently recruited to target gene promoters by balanced translocation derived oncogenic fusion proteins. As important epigenetic effector mechanisms, histone deacetylases (HDAC) have emerged as potential therapeutic targets. However, the patterns of HDAC1 localization and the role of HDACs in leukemia pathogenesis remain to be elucidated. Using ChIP-Chip analyses we analyzed HDAC1 deposition patterns at more than 10,000 gene promoters in a large cohort of leukemia patients and CD34+ controls. HDAC1 binding was significantly increased in AML blasts compared to CD34+ progenitor cells at 130 gene promoters whereas decreased binding was observed at 66 gene promoters. Distinct HDAC1 binding patterns occurred in AML subtypes with balanced translocations t(15;17), t(8;21) and inv(16). In addition, a more generalized signature was established, that revealed an AML specific pattern of HDAC1 distribution. Many of the HDAC1-binding altered promoters regulate genes involved in hematopoiesis, transcriptional regulation and signal transduction. HDAC1 binding patterns were associated with patients' event free survival. This is the first study to determine HDAC1 modification patterns in a large number of AML and ALL specimens. Our findings suggest that dyslocalization of HDAC1 is a common feature in AML. Importantly, HDAC1 modifications possess prognostic power for patient survival. Our findings suggest that altered HDAC1 localization is an explanation for the observed benefit of HDAC inhibitors in AML therapy.

Objective Platelet-derived growth factor receptors α and β (PDGFRA, PDGFRB) are frequently expressed on hematopoietic cells and regulate cellular responses such as proliferation, differentiation, survival, and transformation. Stimulation by autocrine loops or activation by chromosomal translocation makes them important factors in development of hematopoietic disorders. Interaction with the ligand PDGF results in activation of the tyrosine kinase domain and phosphorylation of tyrosine residues, thereby creating binding sites for molecules containing Src homology 2 domains. We hypothesized that one such protein may be Lnk, a negative regulator of cytokine receptors, including Mpl, EpoR, c-Kit, and c-Fms. Materials and Methods Interaction of Lnk with PDGFRA, PDGFRB, or leukemogenic FIP1L1-PDGFRA or TEL-PDGFRB was studied in cotransfected 293T cells. Effects of Lnk on PDGFR signaling were shown in 293T and NIH3T3 cells, whereas its influence on either PDGF-dependent or factor-independent growth was investigated using Ba/F3 or 32D cells expressing wild-type PDGFR, FIP1L1-PDGFRA, or TEL-PDGFRB. Results We show that Lnk binds to PDGFR after exposure of cells to PDGF. Furthermore, Lnk can bind the FIP1L1-PDGFRA fusion protein. Mutation or deletion of the Lnk Src homology 2 domain completely abolished binding of Lnk to FIP1L1-PDGFRA, but just partly prevented binding to PDGFRA or PDGFRB. Expression of Lnk inhibited proliferation of PDGF-dependent Ba/F3 cells and diminished phosphorylation of Erk in PDGF-treated NIH3T3. 32D cells transformed by either FIP1L1-PDGFRA or TEL-PDGFRB stopped growing when Lnk was expressed. Conclusions Lnk is a negative regulator of PDGFR signaling. Development of Lnk mimetic drugs might provide a novel therapeutic strategy for myeloproliferative disorders.

The most frequent translocation t(8;21) in acute myeloid leukemia (AML) generates the chimeric AML1/ETO protein, which blocks differentiation and induces self-renewal in hematopoietic progenitor cells. The underlying mechanisms mediating AML1/ETO-induced self-renewal are largely unknown. Using expression microarray analysis, we identified the Groucho-related amino-terminal enhancer of split (AES) as a consistently up-regulated AML1/ETO target. Elevated levels of AES mRNA and protein were confirmed in AML1/ETO-expressing leukemia cells, as well as in other AML specimens. High expression of AES mRNA or protein was associated with improved survival of AML patients, even in the absence of t(8;21). On a functional level, knockdown of AES by RNAi in AML1/ETO-expressing cell lines inhibited colony formation. Similarly, self-renewal induced by AML1/ETO in primary murine progenitors was inhibited when AES was decreased or absent. High levels of AES expression enhanced formation of immature colonies, serial replating capacity of primary cells, and colony formation in colony-forming unit-spleen assays. These findings establish AES as a novel AML1/ETO-induced target gene that plays an important role in the self-renewal phenotype of t(8;21)-positive AML.

The FIP1L1-PDGFRA fusion is seen in a fraction of cases with a presumptive diagnosis of hypereosinophilic syndrome (HES). However, because most HES patients lack FIP1L1-PDGFRA, we studied whether they harbor activating mutations of the PDGFRA gene. Sequencing of 87 FIP1L1-PDGFRA–negative HES patients revealed several novel PDGFRA point mutations (R481G, L507P, I562M, H570R, H650Q, N659S, L705P, R748G, and Y849S). When cloned into 32D cells, N659S and Y849S and—on selection for high expressors—also H650Q and R748G mutants induced growth factor–independent proliferation, clonogenic growth, and constitutive phosphorylation of PDGFRA and Stat5. Imatinib antagonized Stat5 phosphorylation. Mutations involving positions 659 and 849 had been shown previously to possess transforming potential in gastrointestinal stromal tumors. Because H650Q and R748G mutants possessed only weak transforming activity, we injected 32D cells harboring these mutants or FIP1L1-PDGFRA into mice and found that they induced a leukemia-like disease. Oral imatinib treatment significantly decreased leukemic growth in vivo and prolonged survival. In conclusion, our data provide evidence that imatinib-sensitive PDGFRA point mutations play an important role in the pathogenesis of HES and we propose that more research should be performed to further define the frequency and treatment response of PDGFRA mutations in FIP1L1-PDGFRA–negative HES patients.

AML progenitor cells (AML-PC) undergo significant apoptosis in response to the deacetylase inhibitor (DACi) LAQ824 and lose the replating capacity which was not observed with the DACi valproic acid. Treatment of normal hematopoietic progenitor cells (HPC) with LAQ824 resulted in (i) inhibition of differentiation, (ii) an G2/M cell cycle arrest exclusively in multipotent CD34 + HPC and (iii) induction of apoptosis predominantly in committed CD34 − HPC. Gene expression analysis showed induction of coactivator and target genes of the notch pathway as well as cell cycle arrest-inducing genes in the most primitive CD34 + CD38 − HPC population which may in part be responsible for the considerable, but reversible haematotoxicity of this drug.

Acute myeloid leukemia (AML) is commonly associated with alterations in transcription factors because of altered expression or gene mutations. These changes might induce leukemia-specific patterns of histone modifications. We used chromatin-immunoprecipitation on microarray to analyze histone 3 lysine 9 trimethylation (H3K9me3) patterns in primary AML (n = 108), acute lymphoid leukemia (n = 28), CD34+ cells (n = 21) and white blood cells (n = 15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions. The altered genomic regions showed an overrepresentation of cis-binding sites for ETS and cyclic adenosine monophosphate response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML-specific H3K9me3 patterns were not associated with known cytogenetic abnormalities. But a signature derived from H3K9me3 patterns predicted event-free survival in AML patients. When the H3K9me3 signature was combined with established clinical prognostic markers, it outperformed prognosis prediction based on clinical parameters alone. These findings demonstrate widespread changes of H3K9me3 levels at gene promoters in AML. Signatures of histone modification patterns are associated with patient prognosis in AML.

Antigen receptors transduce complex signals and thereby control important cell fate decisions of the B-lineage including differentiation, proliferation and B-cell survival. The initial steps of B-cell development take place in the bone marrow, and the first dependency of a developing B-cell on antigen receptor signaling occurs at the pre-B-cell stage where expression of the pre-BCR is essential for cell survival. In contrast to B-cell-receptor (BCR) signaling, the nature of pre-BCR signals is far less characterized. It is believed that pre-BCRs transduce a constitutive baseline signal that is essential for survival and differentiation of pre-B-cells into immature B-cells. Since pre-BCRs are crucial regulators of pre-B-cell survival and differentiation, it is not surprising that dysregulation of pre-BCR signaling is observed in acute lymphocytic leukemia (ALL) cells where it contributes to survival of the malignant cell clones. To identify drug targets in the pre-BCR signaling network, we have systematically characterized pre-BCR signaling in two pre-BCR-dependent ALL cell lines by quantitative phosphoproteomics. Because pre-BCRs transduce a constitutive baseline signal, we interfered with pre-BCR signaling by either Syk inhibitor treatment or by an inducible shRNA-mediated knock-down of the pre-BCR component Ig-α to monitor pre-BCR-dependent signals. We identified and quantified more than 15,000 phospho-sites. Among these sites, more than 500 were regulated upon interference with pre-BCR signaling. Although the upstream components are shared between pre-BCRs and BCRs, the wiring of their downstream signals occurred to be different which is reflected by our finding that the majority of the identified pre-BCR signaling effectors have not been described in the context of BCR signaling in mature B-cells or lymphomas. Finally, we have identified vulnerabilities in the pre-BCR signaling network by intersecting our phosphoproteomic data with data from loss-of-function CRISPR-Cas9 screens for essential genes in ALL cell lines. Taken together, this study provides a comprehensive dataset for pre-BCR signaling and identifies effector proteins that are relevant for the regulation of ALL cell survival and proliferation. Citation Format: Roland Walter, Khouloud Kouidri, Jessica Peter, Martine Pape, Laura Merschen, Bjoern Haeupl, Carmen Doebele, Hubert Serve, Yanlong Yi, Henning Urlaub, Thomas Oellerich. Elucidation of the pre-B-cell receptor signaling network in acute lymphocytic leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2692.

Lnk physically interacts with c-Fms and blunts its activity in-cluding proliferation of macrophage progenitor cells, M-CSF stimulated migration, and generaton of ROS. The M-CSFR (c-Fms) participates in proliferation, differentiation, and survival of macrophages and is involved in the regulation of distinct macrophage functions. Interaction with the ligand M-CSF results in phosphorylation of tyrosine residues on c-Fms, thereby creating binding sites for molecules containing SH2 domains. Lnk is a SH2 domain adaptor protein that negatively regulates hematopoietic cytokine receptors. Here, we show that Lnk binds to c-Fms. Biological and functional effects of this interaction were examined in macrophages from Lnk-deficient (KO) and WT mice. Clonogenic assays demonstrated an elevated number of M-CFUs in the bone marrow of Lnk KO mice. Furthermore, the M-CSF-induced phosphorylation of Akt in Lnk KO macrophages was increased and prolonged, whereas phosphorylation of Erk was diminished. Zymosan-stimulated production of ROS was increased dramatically in a M-CSF-dependent manner in Lnk KO macrophages. Lastly, Lnk inhibited M-CSF-induced migration of macrophages. In summary, we show that Lnk binds to c-Fms and can blunt M-CSF stimulation. Modulation of levels of Lnk in macrophages may provide a unique therapeutic approach to increase innate host defenses.

Recent evidence has demonstrated that acquired uniparental disomy (aUPD) is a novel mechanism by which pathogenetic mutations in cancer may be reduced to homozygosity. To help identify novel mutations in myeloproliferative neoplasms (MPNs), we performed a genome-wide single nucleotide polymorphism (SNP) screen to identify aUPD in 58 patients with atypical chronic myeloid leukemia (aCML; n = 30), JAK2 mutation–negative myelofibrosis (MF; n = 18), or JAK2 mutation–negative polycythemia vera (PV; n = 10). Stretches of homozygous, copy neutral SNP calls greater than 20Mb were seen in 10 (33%) aCML and 1 (6%) MF, but were absent in PV. In total, 7 different chromosomes were involved with 7q and 11q each affected in 10% of aCML cases. CBL mutations were identified in all 3 cases with 11q aUPD and analysis of 574 additional MPNs revealed a total of 27 CBL variants in 26 patients with aCML, myelofibrosis or chronic myelomonocytic leukemia. Most variants were missense substitutions in the RING or linker domains that abrogated CBL ubiquitin ligase activity and conferred a proliferative advantage to 32D cells overexpressing FLT3. We conclude that acquired, transforming CBL mutations are a novel and widespread pathogenetic abnormality in morphologically related, clinically aggressive MPNs.

The adaptor protein Lnk is expressed in haemopoietic cells and plays a critical role in haemopoiesis. Animal model studies demonstrated that Lnk acts as a broad inhibitor of signalling pathways in haemopoietic lineages. Lnk belongs to a family of proteins sharing several structural motifs, including an SH2 (Src homology 2) domain which binds phosphotyrosine residues in various signal-transducing proteins. The SH2 domain is essential for Lnk-mediated negative regulation of several cytokine receptors [e.g. Mpl, EpoR (erythropoietin receptor), c-Kit]. Therefore inhibition of the binding of Lnk to cytokine receptors might lead to enhanced downstream signalling of the receptor and thereby to improved haemopoiesis in response to exposure to cytokines (e.g. erythropoietin in anaemic patients). This hypothesis led us to define the exact binding site of Lnk to the stem cell factor receptor c-Kit. Pull-down experiments using GST (glutathione transferase)-fusion proteins of the different domains of c-Kit showed that Lnk almost exclusively binds to the phosphorylated juxtamembrane domain. Binding of Lnk to the juxtamembrane domain was abolished by point mutation of Tyr568 and was competed by peptides with a phosphotyrosine residue at position 568. Co-immunoprecipitation with full-length wild-type or Y568F mutant c-Kit and Lnk confirmed these results, thus showing the importance of this phosphorylated tyrosine residue. Lnk bound directly to c-Kit without requiring other interacting partners. The identification of the binding site of Lnk to c-Kit will be useful to discover inhibitory molecules that prevent the binding of these two proteins, thus making haemopoietic cells more sensitive to growth factors.

The Wnt-signaling pathway plays a critical role in directing cell fate during embryogenesis and also in the pathogenesis of cancer. In leukemia, it is well described that activating internal tandem duplications (ITD) mutations in receptor tyrosine kinases like cKit or Flt3 confer to the pathogenesis of cancer. Here, we analyzed whether Wnt-signaling plays a role in cKit-ITD mediated transformation. Stably transfected 32D cells with cKit-ITD cells had higher beta-Catenin protein levels compared to the cKit-WT. Analysis of beta-Catenin mRNA and protein levels revealed that beta-Catenin was regulated at post-transcriptional level in cKit-ITD as well as Flt3-ITD compared to the wildtype. Signaling analyses revealed higher-phosphorylation of GSK3beta by oncogenic cKit-ITD. Moreover, activation of Wnt signaling was confirmed by constitutive activation of c-myc luciferase by cKit-ITD cells. Importantly, using dominant negative TCF4, we show that activation of Wnt signaling plays an important role in cKit mediated transformation of myeloid cells. Application of specific receptor tyrosine kinase inhibitors for Flt3 or cKit result in a decrease of beta-Catenin that underwent with a decrease of GSK3beta phosphorylation, suggesting an indirect mechanism of beta-Catenin regulation by oncogenic receptor tyrosine kinases in both ITD mutations. Our study shows the importance of activation of Wnt signaling in leukemia and suggests as attractive target for future therapeutical approaches.

Mutations in the receptor tyrosine kinase Flt3 represent one of the most common known genetic lesions in AML. Internal tandem duplication (ITD) mutations clustered in the juxtamembrane domain are the most frequent and best characterized mutations found in Flt3. We and others have previously reported that oncogenic activation of Flt3 by ITD mutations activate aberrant signaling including activation of STAT5, up-regulation of STAT targets like-Pim and SOCS proteins and repression of the myeloid transcription factors Pu.1 and c/EBP-alpha (Mizuki et al., Blood96: 3907–14, 2000; Mizuki et al., Blood101: 3164–73, 2003). Earlier studies have observed activation of Src and Jak kinases by Flt3-ITD but the mechanisms of STAT5 activation by Flt3-ITD and the role of Src or Jak kinases in this process remains unclear. Using the specific small molecule inhibitors PP-1 and PP-2, we analyzed the role of Src kinases in Flt3-ITD mediated STAT5 activation. Treatment of Flt3-ITD expressing 32D cells with PP-1 and 2 had only marginal effect on Flt3-ITD induced STAT5 activation, which was due to a proportional inhibition of Flt3 kinase by these inhibitors. Similar results were obtained with AG490, reportedly a specific inhibitor of Jak2. However, use of cell a line deficient for all Src family kinases revealed a Src-independent activation of STAT5 by Flt3-ITD. Also, using cell lines deficient for Jak2 or Tyk2 we show that Flt3-ITD induced STAT5 activation independent of Jak2 or Tyk2 kinases. To rule out a possible redundant role of different Jak kinases in Flt3-ITD mediated STAT5 activation, we used SOCS1, a target of STAT5 and a potent molecular inhibitor of all Jak kinases. Surprisingly, retroviral transduction of 32D cells with SOCS1 inhibited IL-3 but not Flt3-ITD mediated proliferation of these cells. Similar results were obtained with SOCS3, another inhibitor of Jak kinases. Detailed signaling analyses of stable 32D cells co-expressing Flt3-ITD and SOCS1 revealed that activation of the STAT5 and up-regulation of STAT5 target gene Pim-2 by Flt3-ITD is completely resistant to SOCS1. In contrast, expression of SOCS1 severely inhibited IL-3 mediated STAT5 activation. Furthermore, using purified recombinant STAT5 and Flt3 kinase in in-vitro kinase assays, we show that STAT5 is indeed a direct target of Flt3. Taken together, our data show that Flt3-ITD directly activates STAT5 independent of Src or Jak kinases, providing the mechanistic basis of Flt3-ITD induced STAT5 activation. Furthermore, our finding that Flt3-ITD up-regulates SOCS proteins but activate STAT5 in a SOCS1 resistant manner may have important implications for Flt3-ITD-mediated modification of cytokine signaling.

Epigenetic changes play an important role in leukemia pathogenesis. DNA methylation is among the most common alterations in leukemia. The potential role of DNA methylation as a biomarker in leukemia is unknown. In addition, the lack of molecular markers precludes minimal residual disease (MRD) estimation for most patients with hematologic malignancies. We analyzed the potential of aberrant DNA promoter methylation as a biomarker for MRD in acute leukemias. Quantitative real-time PCR methods with bisulfite-modified DNA were established to quantify MRD based on estrogen receptor α (ERα) and/or p15INK4B methylation. Methylation analyses were done in >370 DNA specimens from 180 acute leukemia patients and controls. Methylation of ERα and/or p15INK4B occurred frequently and specifically in acute leukemia but not in healthy controls or in nonmalignant hematologic diseases. Aberrant DNA methylation was detectable in >20% of leukemia patients during clinical remission. In pediatric acute lymphoblastic leukemia, methylation levels during clinical remission correlated closely with T-cell receptor/immunoglobulin MRD levels (r = +0.7, P < 0.01) and were associated with subsequent relapse. In acute myelogenous leukemia patients in clinical remission, increased methylation levels were associated with a high relapse risk and significantly reduced relapse-free survival (P = 0.003). Many patients with acute leukemia in clinical remission harbor increased levels of aberrant DNA methylation. Analysis of methylation MRD might be used as a novel biomarker for leukemia patients' relapse risk. [Cancer Res 2007;67(3):1370–7]

Cyclin A1 plays a critical role in hematopoietic malignancies, notably, acute myeloid leukemia. The molecular mechanisms of cyclin A1 action are incompletely understood. Here, we show that cyclin A1 functions are mediated by the retinoblastoma and the Ku70 pathway. High levels of cyclin A1 and the associated CDK2 kinase activity were associated with increasing levels of phosphorylated retinoblastoma in vivo. UV irradiation induced a switch of the CDK2 towards cyclin A1, with accordance to changes in CDK2 kinase activity. The C-terminus of cyclin A1 directly interacted with Ku70, and DNA binding activity of Ku70 was modulated by cyclin A1/CDK2 and phosphatase treatment. Cyclin A1-deficiency induced by shRNA increased apoptosis that is induced by DNA damage and death receptor ligands. Taken together, these analyses demonstrate that cyclin A1 exerts antiapoptotic functions by interacting with retinoblastoma and Ku proteins in leukemia cells.

Treatment success in patients with acute myeloid leukaemia (AML) is heterogeneous. Cytogenetic and molecular alterations are strong prognostic factors, which have been used to individualize treatment. Here, we studied the impact of TP53 mutations on the outcome of AML patients with adverse cytogenetic risk treated with allogeneic haematopoietic stem cell transplantation (HSCT). Samples of 97 patients with AML and adverse-risk cytogenetics who had received a HSCT within three randomized trials were analysed. Complete sequencing of the TP53 coding region was performed using next generation sequencing. The median age was 51 years. Overall, TP53 mutations were found in 40 patients (41%). With a median follow up of 67 months, the three-year probabilities of overall survival (OS) and event-free survival for patients with TP53 wild type were 33% [95% confidence interval (CI), 21% to 45%] and 24% (95% CI, 13% to 35%) compared to 10% (95% CI, 0% to 19%) and 8% (95% CI, 0% to 16%) (P = 0·002 and P = 0·007) for those with mutated TP53, respectively. In multivariate analysis, the TP53-mutation status had a negative impact on OS (Hazard Ratio = 1·7; P = 0·066). Mutational analysis of TP53 might be an important additional tool to predict outcome after HSCT in patients with adverse karyotype AML.

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.

The slowing down or stalling of replication forks is commonly known as replication stress and arises from multiple causes such as DNA lesions, nucleotide depletion, RNA-DNA hybrids, and oncogene activation. The ataxia telangiectasia and Rad3-related kinase (ATR) plays an essential role in the cellular response to replication stress and inhibition of ATR has emerged as therapeutic strategy for the treatment of cancers that exhibit high levels of replication stress. However, the cellular signaling induced by replication stress and the substrate spectrum of ATR has not been systematically investigated. In this study, we employed quantitative MS-based proteomics to define the cellular signaling after nucleotide depletion-induced replication stress and replication fork collapse following ATR inhibition. We demonstrate that replication stress results in increased phosphorylation of a subset of proteins, many of which are involved in RNA splicing and transcription and have previously not been associated with the cellular replication stress response. Furthermore, our data reveal the ATR-dependent phosphorylation following replication stress and discover novel putative ATR target sites on MCM6, TOPBP1, RAD51AP1, and PSMD4. We establish that ATR inhibition rewires cellular signaling networks induced by replication stress and leads to the activation of the ATM-driven double-strand break repair signaling.