Senescence-associated reprogramming promotes cancer stemness.

Cellular senescence is a stress-responsive cell-cycle arrest program that terminates the further expansion of (pre-)malignant cells. Key signalling components of the senescence machinery, such as p16INK4a, p21CIP1 and p53, as well as trimethylation of lysine 9 at histone H3 (H3K9me3), also operate as critical regulators of stem-cell functions (which are collectively termed 'stemness'). In cancer cells, a gain of stemness may have profound implications for tumour aggressiveness and clinical outcome. Here we investigated whether chemotherapy-induced senescence could change stem-cell-related properties of malignant cells. Gene expression and functional analyses comparing senescent and non-senescent B-cell lymphomas from Eµ-Myc transgenic mice revealed substantial upregulation of an adult tissue stem-cell signature, activated Wnt signalling, and distinct stem-cell markers in senescence. Using genetically switchable models of senescence targeting H3K9me3 or p53 to mimic spontaneous escape from the arrested condition, we found that cells released from senescence re-entered the cell cycle with strongly enhanced and Wnt-dependent clonogenic growth potential compared to virtually identical populations that had been equally exposed to chemotherapy but had never been senescent. In vivo, these previously senescent cells presented with a much higher tumour initiation potential. Notably, the temporary enforcement of senescence in p53-regulatable models of acute lymphoblastic leukaemia and acute myeloid leukaemia was found to reprogram non-stem bulk leukaemia cells into self-renewing, leukaemia-initiating stem cells. Our data, which are further supported by consistent results in human cancer cell lines and primary samples of human haematological malignancies, reveal that senescence-associated stemness is an unexpected, cell-autonomous feature that exerts its detrimental, highly aggressive growth potential upon escape from cell-cycle blockade, and is enriched in relapse tumours. These findings have profound implications for cancer therapy, and provide new mechanistic insights into the plasticity of cancer cells.

Different treatment strategies versus a common standard arm (CSA) in patients with newly diagnosed AML over the age of 60 years: a randomized German inter-group study.

A randomized inter-group trial comparing more intensive treatment strategies to a common standard arm 3 + 7 (CSA) was conducted in patients with non-M3 AML. Untreated patients ≥ 60 years were allocated to the CSA (n = 132) or to the study group arms (n = 1154) of the AMLCG (TAD/HAM versus HAM/HAM ± G-CSF followed by TAD and maintenance) and the OSHO (intermediate-dose ara-C/mitoxantrone followed by ara-C/mitoxantrone). Median age of the 1147 eligible patients was 69 (range 60-87) years. CR/CRi status at 90 days was not significantly different between the CSA (54% (95%CI: 45-64)) and the study group arms (53% (95%CI: 47-60) and 59% (95%CI: 58-63)). The five-year event-free survival (EFS) probability (primary endpoint) was 6.2% (95%CI: 2.7-14.0) in the CSA, 7.6% (95%CI: 4.5-12.8) in study group A and 11.1% (95%CI: 9.0-13.7) in B. The 5-year OS was 17.2% (95%CI: 11.0-26.9), 17.0% (95%CI: 2.0-23.9), and 19.5% (95%CI: 16.7-22.8) in CSA, study group A and B, respectively. Neither study group differed significantly from the CSA regarding EFS, OS, or relapse-free survival. In multivariate analyses, allocation to the treatment strategy was not significantly associated with the time-to-event endpoints. The evaluation of more intensive treatment strategies did not show clinically relevant outcome differences when compared to CSA.

The sorting receptor Sortilin exhibits a dual function in exocytic trafficking of interferon-γ and granzyme A in T cells.

Immunological control of infections or tumors depends on the release of effector cytokines and polarized secretion of cytotoxic granules from T cells and natural killer cells. Here we show that the sorting receptor Sortilin controlled both processes. In murine Sortilin-deficient cytotoxic T lymphocytes, regulated secretion of granzyme A and cytotoxic killing was enhanced and correlated with increased vesicle-associated membrane protein 7 availability. In contrast, loss of Sortilin reduced the release of interferon-γ upon infections and in autoimmune colitis. Exit of interferon-γ from the Golgi apparatus required the presence of Sortilin. Furthermore, we tracked the transport route of interferon-γ beyond this Sortilin-dependent Golgi to early endosome step. In wild-type T cells, trafficking of interferon-γ from the endosomal sorting platform to the plasma membrane proceeded independently of recycling endosomes, and interferon-γ remained excluded from late endosomes. Our results suggest that Sortilin modulates systemic immune responses through exocytic sorting of immunological effector molecules.

Pharmacological restoration and therapeutic targeting of the B-cell phenotype in classical Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL), although originating from B cells, is characterized by the virtual lack of gene products whose expression constitutes the B-cell phenotype. Epigenetic repression of B-cell-specific genes via promoter hypermethylation and histone deacetylation as well as compromised expression of B-cell-committed transcription factors were previously reported to contribute to the lost B-cell phenotype in cHL. Restoring the B-cell phenotype may not only correct a central malignant property, but it may also render cHL susceptible to clinically established antibody therapies targeting B-cell surface receptors or small compounds interfering with B-cell receptor signaling. We conducted a high-throughput pharmacological screening based on >28 000 compounds in cHL cell lines carrying a CD19 reporter to identify drugs that promote reexpression of the B-cell phenotype. Three chemicals were retrieved that robustly enhanced CD19 transcription. Subsequent chromatin immunoprecipitation-based analyses indicated that action of 2 of these compounds was associated with lowered levels of the transcriptionally repressive lysine 9-trimethylated histone H3 mark at the CD19 promoter. Moreover, the antileukemia agents all-trans retinoic acid and arsenic trioxide (ATO) were found to reconstitute the silenced B-cell transcriptional program and reduce viability of cHL cell lines. When applied in combination with a screening-identified chemical, ATO evoked reexpression of the CD20 antigen, which could be further therapeutically exploited by enabling CD20 antibody-mediated apoptosis of cHL cells. Furthermore, restoration of the B-cell phenotype also rendered cHL cells susceptible to the B-cell non-Hodgkin lymphoma-tailored small-compound inhibitors ibrutinib and idelalisib. In essence, we report here a conceptually novel, redifferentiation-based treatment strategy for cHL.

Synthetic lethal metabolic targeting of cellular senescence in cancer therapy.

Activated oncogenes and anticancer chemotherapy induce cellular senescence, a terminal growth arrest of viable cells characterized by S-phase entry-blocking histone 3 lysine 9 trimethylation (H3K9me3). Although therapy-induced senescence (TIS) improves long-term outcomes, potentially harmful properties of senescent tumour cells make their quantitative elimination a therapeutic priority. Here we use the Eµ-myc transgenic mouse lymphoma model in which TIS depends on the H3K9 histone methyltransferase Suv39h1 to show the mechanism and therapeutic exploitation of senescence-related metabolic reprogramming in vitro and in vivo. After senescence-inducing chemotherapy, TIS-competent lymphomas but not TIS-incompetent Suv39h1(-) lymphomas show increased glucose utilization and much higher ATP production. We demonstrate that this is linked to massive proteotoxic stress, which is a consequence of the senescence-associated secretory phenotype (SASP) described previously. SASP-producing TIS cells exhibited endoplasmic reticulum stress, an unfolded protein response (UPR), and increased ubiquitination, thereby targeting toxic proteins for autophagy in an acutely energy-consuming fashion. Accordingly, TIS lymphomas, unlike senescence models that lack a strong SASP response, were more sensitive to blocking glucose utilization or autophagy, which led to their selective elimination through caspase-12- and caspase-3-mediated endoplasmic-reticulum-related apoptosis. Consequently, pharmacological targeting of these metabolic demands on TIS induction in vivo prompted tumour regression and improved treatment outcomes further. These findings unveil the hypercatabolic nature of TIS that is therapeutically exploitable by synthetic lethal metabolic targeting.

Opposing roles of NF-κB in anti-cancer treatment outcome unveiled by cross-species investigations.

In malignancies, enhanced nuclear factor-κB (NF-κB) activity is largely viewed as an oncogenic property that also confers resistance to chemotherapy. Recently, NF-κB has been postulated to participate in a senescence-associated and possibly senescence-reinforcing cytokine response, thereby suggesting a tumor-restraining role for NF-κB. Using a mouse lymphoma model and analyzing transcriptome and clinical data from lymphoma patients, we show here that therapy-induced senescence presents with and depends on active NF-κB signaling, whereas NF-κB simultaneously promotes resistance to apoptosis. Further characterization and genetic engineering of primary mouse lymphomas according to distinct NF-κB-related oncogenic networks reminiscent of diffuse large B-cell lymphoma (DLBCL) subtypes guided us to identify Bcl2-overexpressing germinal center B-cell-like (GCB) DLBCL as a clinically relevant subgroup with significantly superior outcome when NF-κB is hyperactive. Our data illustrate the power of cross-species investigations to functionally test genetic mechanisms in transgenic mouse tumors that recapitulate distinct features of the corresponding human entity, and to ultimately use the mouse model-derived genetic information to redefine novel, clinically relevant patient subcohorts.

Frequent NFKBIE deletions are associated with poor outcome in primary mediastinal B-cell lymphoma.

We recently reported a truncating deletion in the NFKBIE gene, which encodes IκBε, a negative feedback regulator of NF-κB, in clinically aggressive chronic lymphocytic leukemia (CLL). Because preliminary data indicate enrichment of NFKBIE aberrations in other lymphoid malignancies, we screened a large patient cohort (n = 1460) diagnosed with different lymphoid neoplasms. While NFKBIE deletions were infrequent in follicular lymphoma, splenic marginal zone lymphoma, and T-cell acute lymphoblastic leukemia (<2%), slightly higher frequencies were seen in diffuse large B-cell lymphoma, mantle cell lymphoma, and primary central nervous system lymphoma (3% to 4%). In contrast, a remarkably high frequency of NFKBIE aberrations (46/203 cases [22.7%]) was observed in primary mediastinal B-cell lymphoma (PMBL) and Hodgkin lymphoma (3/11 cases [27.3%]). NFKBIE-deleted PMBL patients were more often therapy refractory (P = .022) and displayed inferior outcome compared with wild-type patients (5-year survival, 59% vs 78%; P = .034); however, they appeared to benefit from radiotherapy (P =022) and rituximab-containing regimens (P = .074). NFKBIE aberrations remained an independent factor in multivariate analysis (P = .003) and when restricting the analysis to immunochemotherapy-treated patients (P = .008). Whole-exome sequencing and gene expression profiling verified the importance of NF-κB deregulation in PMBL. In summary, we identify NFKBIE aberrations as a common genetic event across B-cell malignancies and highlight NFKBIE deletions as a novel poor-prognostic marker in PMBL.

C/EBPbetaDeltauORF mice--a genetic model for uORF-mediated translational control in mammals.

Upstream ORFs (uORFs) are translational control elements found predominantly in transcripts of key regulatory genes. No mammalian genetic model exists to experimentally validate the physiological relevance of uORF-regulated translation initiation. We report that mice deficient for the CCAAT/enhancer-binding protein beta (C/EBPbeta) uORF initiation codon fail to initiate translation of the autoantagonistic LIP (liver inhibitory protein) C/EBPbeta isoform. C/EBPbeta(DeltauORF) mice show hyperactivation of acute-phase response genes, persistent repression of E2F-regulated genes, delayed and blunted S-phase entry of hepatocytes after partial hepatectomy, and impaired osteoclast differentiation. These data and the widespread prevalence of uORFs in mammalian transcriptomes suggest a comprehensive role of uORF-regulated translation in (patho)physiology.

Essential role of IRF4 and MYC signaling for survival of anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) is a distinct entity of T-cell lymphoma that can be divided into 2 subtypes based on the presence of translocations involving the ALK gene (ALK(+) and ALK(-) ALCL). The interferon regulatory factor 4 (IRF4) is known to be highly expressed in both ALK(+) and ALK(-) ALCLs. However, the role of IRF4 in the pathogenesis of these lymphomas remains unclear. Here we show that ALCLs of both subtypes are addicted to IRF4 signaling, as knockdown of IRF4 by RNA interference was toxic to ALCL cell lines in vitro and in ALCL xenograft mouse models in vivo. Gene expression profiling after IRF4 knockdown demonstrated a significant downregulation of a variety of known MYC target genes. Furthermore, our analyses revealed that MYC is a primary target of IRF4, identifying a novel regulatory mechanism of MYC expression and its target gene network in ALCL. MYC, itself, is essential for ALCL survival, as both knockdown of MYC and pharmacologic inhibition of MYC signaling were toxic to ALCL cell lines. Collectively, our results demonstrate that ALCLs are dependent on IRF4 and MYC signaling and that MYC may represent a promising target for future therapies.

Predictive significance of the European LeukemiaNet classification of genetic aberrations in patients with acute myeloid leukaemia undergoing allogeneic stem cell transplantation.

OBJECTIVES: The purpose of this study was to evaluate the predictive capacity of the European LeukemiaNet (ELN) classification of genetic risk in patients with acute myeloid leukaemia (AML) undergoing allogeneic stem cell transplantation (alloSCT). METHODS: We retrospectively analysed 274 patients transplanted at our centre between 2004 and 2014. RESULTS: The ELN grouping is comparable to the Southwest Oncology Group/Eastern Cooperative Oncology Group (SWOG/ECOG) stratification in predicting the outcome after alloSCT [overall P = 0.0064 for disease-free survival (DFS), overall P = 0.003 for relapse]. Patients with an intermediate-1 profile have a significantly elevated 5-yr relapse incidence as compared to favourable risk patients, that is 40% vs. 15%, [hazard ratio (HR) 2.58, P = 0.048]. An intermediate-1 risk profile is an independent predictor for relapse as determined by multivariate Cox regression analysis (HR 3.05, P = 0.023). In intermediate-1 patients, the presence of an FLT3 internal tandem duplication (FLT3-ITD) is associated with a significantly increased relapse incidence (P = 0.0323), and a lower DFS (P = 0.0465). FLT3-ITD is an independent predictor for overall survival, DFS and relapse incidence in the intermediate-1 subgroup. CONCLUSIONS: The ELN stratification of genetic risk predicts the outcome of patients with AML undergoing alloSCT. Patients with an intermediate-1 profile have a high risk for treatment failure due to relapse, which prompts the development of alternative treatment strategies.

Mapping of transcription factor motifs in active chromatin identifies IRF5 as key regulator in classical Hodgkin lymphoma.

Deregulated transcription factor (TF) activities are commonly observed in hematopoietic malignancies. Understanding tumorigenesis therefore requires determining the function and hierarchical role of individual TFs. To identify TFs central to lymphomagenesis, we identified lymphoma type-specific accessible chromatin by global mapping of DNaseI hypersensitive sites and analyzed enriched TF-binding motifs in these regions. Applying this unbiased approach to classical Hodgkin lymphoma (HL), a common B-cell-derived lymphoma with a complex pattern of deregulated TFs, we discovered interferon regulatory factor (IRF) sites among the top enriched motifs. High-level expression of the proinflammatory TF IRF5 was specific to HL cells and crucial for their survival. Furthermore, IRF5 initiated a regulatory cascade in human non-Hodgkin B-cell lines and primary murine B cells by inducing the TF AP-1 and cooperating with NF-κB to activate essential characteristic features of HL. Our strategy efficiently identified a lymphoma type-specific key regulator and uncovered a tumor promoting role of IRF5.

Multiple myeloma cells modify VEGF/IL-6 levels and osteogenic potential of bone marrow stromal cells via Notch/miR-223.

Bone marrow mesenchymal stromal cells (BMMSCs) represent a crucial component of multiple myeloma (MM) microenvironment supporting its progression and proliferation. Recently, microRNAs have become an important point of interest for research on micro-environmental interactions in MM with some evidence of tumor supportive roles in MM. In this study, we examined the role of miR-223 for MM support in BMMSCs of 56 patients with MM (MM-BMMSCs). miR-223 expression in MM-BMMSCs was reduced by the presence of MM cells in vitro in a cell-contact dependent manner compared to mono-cultured MM-BMMSCs. Co-cultivation of MM cells and MM-BMMSCs induced activation of notch amongst others via jagged-2/notch-2 leading to increased expression of Hes1, Hey2, or Hes5 in both cell types. Cultivation of MM-BMMSCs with increasing levels of recombinant jagged-2 reduced miR-223 and increased Hes1 levels in a concentration-dependent manner. Transient reduction of miR-223 levels increased VEGF and IL-6 expression and secretion by MM-BMMSCs. In addition, reduction of miR-223 degraded the osteogenic differentiation potential of MM-BMMSCs. Inhibition of notch signaling induced apoptosis in both MM cells and MM-BMMSCs. Furthermore, it increased miR-223 levels and reduced expression of VEGF and IL-6 by both cell types. These data provide first evidence that miR-223 participates in different MM supporting pathways in MM-BMMSCs inlcuding regulation of cytokine secretion and expression as well as osteogenic differentiation of MM-BMMSCs. More insights on the role of miR-223 in MM-BMMSCs and in cellular interactions between MM cells and MM-BMMSCs could provide starting points for a more efficient anti-myeloma treatment by targeting of notch signaling. © 2015 Wiley Periodicals, Inc.

Bone marrow T-cell infiltration during acute GVHD is associated with delayed B-cell recovery and function after HSCT.

B-cell immune dysfunction contributes to the risk of severe infections after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Delayed B-cell regeneration is found in patients with systemic graft-versus-host disease (GVHD) and is often accompanied by bone marrow (BM) suppression. Little is known about human BM GVHD. We analyzed the reconstitution kinetics of B-cell subsets in adult leukemic patients within 6 months after allo-HSCT. B-cell deficiency already existed before transplant and was aggravated after transplant. Onset of B-cell reconstitution characterized by transitional B-cell recovery occurred either early (months 2-3) or late (from month 6 on) and correlated highly positively with reverse transcription-polymerase chain reaction quantified numbers of κ-deleting recombination excision circles (KRECs). Delayed recovery was associated with systemic acute GVHD and full-intensity conditioning therapy. Histological analysis of BM trephines revealed increased T-cell infiltration in late recovering patients, which was associated with reduced numbers of osteoblasts. Functionally, late recovering patients displayed less pneumococcal polysaccharide-specific immunoglobin M-producing B cells on ex vivo B-cell activation than early recovering patients. Our results provide evidence for acute BM GVHD in allo-HSCT patients with infiltrating donor T cells and osteoblast destruction. This is associated with delayed B-cell reconstitution and impaired antibody response. Herein, KREC appears suitable to monitor BM B-cell output after transplant.

BCR-ABL-positive acute myeloid leukemia: a new entity? Analysis of clinical and molecular features.

BCR-ABL-positive acute myeloid leukemia (AML) is a rare subtype of AML that is now included as a provisional entity in the 2016 revised WHO classification of myeloid malignancies. Since a clear distinction between de novo BCR-ABL+ AML and chronic myeloid leukemia (CML) blast crisis is challenging in many cases, the existence of de novo BCR-ABL+ AML has been a matter of debate for a long time. However, there is increasing evidence suggesting that BCR-ABL+ AML is in fact a distinct subgroup of AML. In this study, we analyzed all published cases since 1975 as well as cases from our institution in order to present common clinical and molecular features of this rare disease. Our analysis shows that BCR-ABL predominantly occurs in AML-NOS, CBF leukemia, and AML with myelodysplasia-related changes. The most common BCR-ABL transcripts (p190 and p210) are nearly equally distributed. Based on the analysis of published data, we provide a clinical algorithm for the initial differential diagnosis of BCR-ABL+ AML. The prognosis of BCR-ABL+ AML seems to depend on the cytogenetic and/or molecular background rather than on BCR-ABL itself. A therapy with tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib, or nilotinib is reasonable, but-due to a lack of systematic clinical data-their use cannot be routinely recommended in first-line therapy. Beyond first-line treatment of AML, the use of TKI remains an individual decision, both in combination with intensive chemotherapy and/or as a bridge to allogeneic stem cell transplantation. In each single case, potential benefits have to be weighed against potential risks.

PTEN loss defines a PI3K/AKT pathway-dependent germinal center subtype of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) represents a heterogeneous diagnostic category with distinct molecular subtypes that can be defined by gene expression profiling. However, even within these defined subtypes, heterogeneity prevails. To further elucidate the pathogenesis of these entities, we determined the expression of the tumor suppressor phosphatase and tensin homolog (PTEN) in 248 primary DLBCL patient samples. These analyses revealed that loss of PTEN was detectable in 55% of germinal center B-cell-like (GCB) DLBCLs, whereas this abnormality was found in only 14% of non-GCB DLBCL patient samples. In GCB DLBCL, the PTEN status was inversely correlated with activation of the oncogenic PI3K/protein kinase B (AKT) pathway in both DLBCL cell lines and primary patient samples. Reexpression of PTEN induced cytotoxicity in PTEN-deficient GCB DLBCL cell line models by inhibiting PI3K/AKT signaling, indicating an addiction to this pathway in this subset of GCB DLBCLs. PI3K/AKT inhibition induced down-regulation of the transcription factor MYC. Reexpression of MYC rescued GCB DLBCL cells from PTEN-induced toxicity, identifying a regulatory mechanism of MYC expression in DLBCL. Finally, pharmacologic PI3K inhibition resulted in toxicity selectively in PTEN-deficient GCB DLBCL lines. Collectively, our results indicate that PTEN loss defines a PI3K/AKT-dependent GCB DLBCL subtype that is addicted to PI3K and MYC signaling and suggest that pharmacologic inhibition of PI3K might represent a promising therapeutic approach in these lymphomas.

Gene expression profiling of peripheral blood mononuclear cells during treatment with a gene-modified allogeneic tumor cell vaccine in advanced renal cell cancer: tumor-induced immunosuppression and a possible role for NF-κB.

Tumor-induced immunosuppression remains a major challenge for immunotherapy of cancer patients. To further elucidate why an allogeneic gene-modified [interleukin-7 (IL-7)/CD80-cotransfected] renal cell cancer (RCC) vaccine failed to induce clinically relevant TH-1-polarized immune responses, peripheral blood mononuclear cells from enrolled study patients were analyzed by gene expression profiling (GEP) both prior and after vaccination. At baseline before vaccination, a profound downregulation of gene signatures associated with antigen presentation, immune response/T cells, cytokines/chemokines and signaling/transcription factors was observed in RCC patients as compared to healthy controls. Vaccination led to a partial reversion of preexisting immunosuppression, however, GEP indicated that an appropriate TH-1 polarization could not be achieved. Most interestingly, our results suggest that the nuclear factor-kappa B signaling pathway might be involved in the impairment of immunological responsiveness and the observed TH-2 deviation. In summary, our data suggest that GEP might be a powerful tool for the prediction of immunosuppression and the monitoring of immune responses within immunotherapy trials.

Apoptosis Susceptibility Prolongs the Lack of Memory B Cells in Acute Leukemic Patients After Allogeneic Hematopoietic Stem Cell Transplantation.

Long-term survival after allogeneic hematopoietic stem cell transplantation requires intact immunosurveillance, which is hampered by lymphoid organ damage associated with conditioning therapy, graft-versus-host disease, and immunosuppression. Our study aimed to identify the mechanisms contributing to sustained low memory B cell numbers after transplantation. Peripheral B and T cell subset recovery and functional marker expression were investigated in 35 acute leukemic patients up to 1 year after transplantation. Apoptosis of B cells after CD40/TLR-9, CD40/BCR, and CD40/BCR/TLR-9-dependent stimulation and drug efflux capacity were analyzed. One half of the patients suffered from infections after day 180. All patients had strongly diminished CD27(+) memory B cells despite already normalized total B cell numbers and fully recovered CD27(-)IgD(-) memory B cells, putatively of extra-follicular origin. Circulating memory follicular helper T cells were reduced in the majority of patients as well. Naïve B cells exhibited a decreased expression of CXCR5, which mediates follicular B cell entry. Additionally, a lower HLA-DR expression was found on naïve B cells, impairing antigen presentation. Upon CD40/TLR-9-dependent activation, B cells underwent significantly increased apoptosis paralleled by an aberrant up-regulation of Fas-L on activated T cells and Fas on resting B cells. Significantly increased B cell apoptosis was also observed after CD40/BCR and CD40/BCR/TLR-9-dependent activation. Drug efflux capacity of naïve B cells was diminished in cyclosporin A-treated patients, additionally contributing to an apoptosis-prone phenotype. We conclude that B cell survival and migration and T cell communication defects are contributing candidates for an impaired germinal center formation of memory B cells after allogeneic hematopoietic stem cell transplantation. Follow-up studies should evaluate effectiveness of revaccinations on the cellular level and should address the long-term sequelae of B cell defects after transplantation.

IκB-ζ controls the constitutive NF-κB target gene network and survival of ABC DLBCL.

Constitutive activation of the nuclear factor-κ B (NF-κB) pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Recurrent mutations of NF-κB regulators that cause constitutive activity of this oncogenic pathway have been identified. However, it remains unclear how specific target genes are regulated. We identified the atypical nuclear IκB protein IκB-ζ to be upregulated in ABC compared with germinal center B-cell-like (GCB) DLBCL primary patient samples. Knockdown of IκB-ζ by RNA interference was toxic to ABC but not to GCB DLBCL cell lines. Gene expression profiling after IκB-ζ knockdown demonstrated a significant downregulation of a large number of known NF-κB target genes, indicating an essential role of IκB-ζ in regulating a specific set of NF-κB target genes. To further investigate how IκB-ζ mediates NF-κB activity, we performed immunoprecipitations and detected a physical interaction of IκB-ζ with both p50 and p52 NF-κB subunits, indicating that IκB-ζ interacts with components of both the canonical and the noncanonical NF-κB pathway in ABC DLBCL. Collectively, our data demonstrate that IκB-ζ is essential for nuclear NF-κB activity in ABC DLBCL, and thus might represent a promising molecular target for future therapies.

Flow cytometric maturity score as a novel prognostic parameter in patients with acute myeloid leukemia.

The European LeukemiaNet (ELN) classification is widely accepted for risk stratification of patients with acute myeloid leukemia (AML). In order to establish immunophenotypic features that predict prognosis, the expression of single AML blast cell antigens has been evaluated with partly conflicting results; however, the influence of immunophenotypic blast maturity is largely unknown. In our study, 300 AML patients diagnosed at our institution between January 2003 and April 2012 were analyzed. A flow cytometric maturity score was developed in order to distinguish "mature" AML (AML-ma) from "immature" AML (AML-im) by quantitative expression levels of early progenitor cell antigens (CD34, CD117, and TdT). AML-ma showed significantly longer relapse-free survival (RFS) and overall survival (OS) than AML-im (p < 0.001). Interestingly, statistically significant differences in RFS and OS were maintained within the "intermediate-risk" group according to ELN (RFS, 7.0 years (AML-ma) vs. 3.3 years (AML-im); p = 0.002; OS, 5.1 years (AML-ma) vs. 3.0 years (AML-im); p = 0.022). Our novel flow cytometric score easily determines AML blast maturity and can predict clinical outcome. It remains to be clarified whether these results simply reflect an accumulation of favorable molecular phenotypes in the AML-ma subgroup or whether they rely on biological differences such as a higher proportion of leukemia stem cells and/or a higher degree of genetic instability within the AML-im subgroup.