C/EBPα deregulation as a paradigm for leukemogenesis.

Myeloid master regulator CCAAT enhancer-binding protein alpha (C/EBPα) is deregulated by multiple mechanisms in leukemia. Inhibition of C/EBPα function plays pivotal roles in leukemogenesis. While much is known about how C/EBPα orchestrates granulopoiesis, our understanding of molecular transformation events, the role(s) of cooperating mutations and clonal evolution during C/EBPα deregulation in leukemia remains elusive. In this review, we will summarize the latest research addressing these topics with special emphasis on CEBPA mutations. We conclude by describing emerging therapeutic strategies to restore C/EBPα function.

Favorable outcome in children and adolescents with a high proportion of advanced phase disease using single/multiple autologous or matched/mismatched allogeneic stem cell transplantations.

We determined the indication, outcome, and risk factors of single and multiple hematopoietic stem cell transplantation(s) (HSCT) in children and adolescents mostly with advanced disease. Forty-one out of 483 patients (8.5 %; median age 9 years) diagnosed at the University of Leipzig with hematological and oncological diseases required HSCT from 1999 to 2011. Patients had overall survival (OS) of 63 ± 10 and 63 ± 16 %, event-free survival (EFS) of 57 ± 10 and 42 ± 16 %, relapse incidence (RI) of 39 ± 10 and 44 ± 18 % and nonrelapse mortality (NRM) of 4 ± 4 and 13 ± 9 % at 10 years after one or more allogeneic and autologous HSCT, respectively. One patient in CR1 and five with advanced disease received two HSCT. Four of the six patients maintained/achieved CR for a median of 13 months. Three died of progression and one of NRM. Two patients had a third HSCT and one survived in CR +231 days after HSCT. Risk factors for OS and EFS were disease stage at HSCT and EBMT risk score. Center (pediatric or JACIE accredited pediatric/adult) was not a determinant for survival. Pediatric single and multiple HSCT are important curative approaches for high-risk malignant diseases with low NRM. Efforts to reduce high RI remain the major aim.

Comparable outcome after single-antigen-mismatched versus matched unrelated donor haematopoietic cell transplantation.

PURPOSE: Allogeneic haematopoietic stem cell transplantation (HSCT) is a proven treatment for patients with haematological malignancies. In this retrospective analysis, the impact of donor matching on outcome of unrelated HSCT was analysed in patients transplanted at the University of Leipzig. METHODS: From 2000 to 2009, 206 patients were transplanted from unrelated donors, of which 51 were mismatched (39 in 1 and 12 in ≥ 2 HLA-antigens), using peripheral blood or bone marrow grafts after total body irradiation and cyclophosphamide or busulfan and cyclophosphamide preparative regimens in combination with ATG. For graft-versus-host disease (GvHD) prophylaxis cyclosporine and MTX were administered. RESULTS: After a median follow-up of 49 months, outcome at 5 years in recipients of HLA-identical grafts was comparable to that of patients transplanted from HLA-incompatible donors with an overall survival (OS) of 52 % (95 % CI 43-61) versus 48 % (95 % CI 34-63), respectively (p = 0.48). Results were also comparable for event-free survival at 5 years [47 % (95 % CI 38-56) vs. 39 % (95 % CI 25-54); p = 0.44], relapse incidence (RI) [29 % (95 % CI 20-38) vs. 41 (95 % CI 25-57); p = 0.22] and non-relapse mortality [24 % (95 % CI 16-33) vs. 20 % (95 % CI 8-33); p = 0.84] in the matched versus mismatched groups. Incidence of acute and chronic GvHD was similar in both groups. Advanced disease (p = 0.02) and low-resolution typing (p = 0.04) are risk factors for OS and RI in univariate and multivariate analysis. CONCLUSIONS: Donors with one antigen mismatch are an acceptable option for patients with malignant disease for whom no fully matched donor is available.

NF-κB/STAT5/miR-155 network targets PU.1 in FLT3-ITD-driven acute myeloid leukemia.

Almost 30% of all acute myeloid leukemias (AML) are associated with an internal tandem duplication (ITD) in the juxtamembrane domain of FMS-like tyrosine kinase 3 receptor (FLT3). Patients with FLT3-ITD mutations tend to have a poor prognosis. MicroRNAs (miRNAs) have a pivotal role in myeloid differentiation and leukemia. MiRNA-155 (MiR-155) was found to be upregulated in FLT3-ITD-associated AMLs. In this study, we discovered that FLT3-ITD signaling induces the oncogenic miR-155. We show in vitro and in vivo that miR-155 expression is regulated by FLT3-ITD downstream targets nuclear factor-κB (p65) and signal transducer and activator of transcription 5 (STAT5). Further, we demonstrate that miR-155 targets the myeloid transcription factor PU.1. Knockdown of miR-155 or overexpression of PU.1 blocks proliferation and induces apoptosis of FLT3-ITD-associated leukemic cells. Our data demonstrate a novel network in which FLT3-ITD signaling induces oncogenic miR-155 by p65 and STAT5 in AML, thereby targeting transcription factor PU.1.

Allogeneic hematopoietic cell transplantation for myelofibrosis in patients pretreated with the JAK1 and JAK2 inhibitor ruxolitinib.

The Janus-activated kinase 1 (JAK1) and JAK2 inhibitor ruxolitinib is effective in decreasing symptomatic splenomegaly and myelofibrosis (MF)-related symptoms. However, allogeneic hematopoietic cell transplantation (HCT) remains the only curative option. We evaluated the impact of ruxolitinib on the outcome after HCT. A cohort of 14 patients (median age 58 years) received a subsequent graft from related (n=3) and unrelated (n=11) donors after a median exposure of 6.5 months to ruxolitinib. At HCT, MF risk for survival according to the International Prognostic Scoring System was intermediate-2 or high risk in 86% of patients. Under ruxolitinib, MF-related symptoms were ameliorated in 10 (71.4%) patients and the palpable spleen reduced by a median of 41% in 7 (64%) of 11 patients with splenomegaly. Engraftment occurred in 13 (93%) patients. Acute GvHD grade-III occurred in 2 (14%) patients. Median follow-up was 9 months. Survival, EFS and treatment-related mortality were 78.6, 64 and 7%, respectively. Through the anti-JAK-mediated reduction in both cytokines and splenomegaly as well as improvement in performance status, ruxolitinib might improve outcome after allogeneic HCT in patients with MF. The downregulation of inflammatory cytokines might have a beneficial impact on graft failure and acute GvHD.

Risk-adapted, treosulfan-based therapy with auto- and allo-SCT for relapsed/refractory aggressive NHL: a prospective phase-II trial.

Since the outcome of relapsed/refractory aggressive non-Hodgkin's lymphoma (NHL) is highly variable, a risk-adapted treatment approach was evaluated. After two cycles of DHAP, patients received high-dose treosulfan/etoposide/carboplatinum (TEC) and autologous stem cell rescue. After TEC, low-risk patients with late relapse (>1 year after first CR who achieved CR after DHAP received no further treatment. Patients with late relapse who achieved CR or PR only after TEC underwent a second cycle of TEC. High-risk patients with early relapse/refractory disease received treosulfan/fludarabine followed by allogeneic transplantation. Rituximab was added in patients with B-cell lymphoma (86%). At entry, 36% of all 57 patients had refractory disease, 32% early and 32% late relapse. During DHAP treatment, progression occurred in 32% of patients. Of 33 patients who received TEC, 5 received second TEC and 15 allogeneic transplantation. Main toxicity after TEC was oral mucositis (CTC grades 3 and 4 in 50% and 13%, respectively). In total, 42% patients achieved CR. Median OS was 21.4 months for all patients and 32.6 for those who underwent allogeneic transplantation. International prognostic index (IPI) at study entry was highly discriminative at predicting OS (P<0.0001). Risk-adapted, treosulfan-based therapy with auto- and allo-SCT is feasible. Long-term survival is possible with allogeneic transplantation.

E6AP, an E3 ubiquitin ligase negatively regulates granulopoiesis by targeting transcription factor C/EBPα for ubiquitin-mediated proteasome degradation.

CCAAT/enhancer-binding protein alpha (C/EBPα) is an important transcription factor involved in granulocytic differentiation. Here, for the first time we demonstrate that E6-associated protein (E6AP), an E3 ubiquitin ligase targets C/EBPα for ubiquitin-mediated proteasome degradation and thereby negatively modulates its functions. Wild-type E6AP promotes ubiquitin dependent proteasome degradation of C/EBPα, while catalytically inactive E6-associated protein having cysteine replaced with alanine at amino-acid position 843 (E6AP-C843A) rather stabilizes it. Further, these two proteins physically associate both in non-myeloid (overexpressed human embryonic kidney epithelium) and myeloid cells. We show that E6AP-mediated degradation of C/EBPα protein expression curtails its transactivation potential on its target genes. Noticeably, E6AP degrades both wild-type 42 kDa CCAAT-enhancer-binding protein alpha (p42C/EBPα) and mutant isoform 30 kDa CCAAT-enhancer-binding protein alpha (p30C/EBPα), this may explain perturbed p42C/EBPα/p30C/EBPα ratio often observed in acute myeloid leukemia (AML). We show that overexpression of catalytically inactive E6AP-C843A in C/EBPα inducible K562- p42C/EBPα-estrogen receptor (ER) cells inhibits ß-estradiol (E2)-induced C/EBPα degradation leading to enhanced granulocytic differentiation. This enhanced granulocytic differentiation upon E2-induced activation of C/EBPα in C/EBPα stably transfected cells (ß-estradiol inducible K562 cells stably expressing p42C/EBPα-ER (K562-C/EBPα-p42-ER)) was further substantiated by siE6AP-mediated knockdown of E6AP in both K562-C/EBPα-p42-ER and 32dcl3 (32D clone 3, a cell line widely used model for in vitro study of hematopoietic cell proliferation, differentiation, and apoptosis) cells. Taken together, our data suggest that E6AP targeted C/EBPα protein degradation may provide a possible explanation for both loss of expression and/or functional inactivation of C/EBPα often experienced in myeloid leukemia.

Human herpesvirus-6 as an inducer of porphyria cutanea tarda: implications from a case.

Here we describe a case that might deliver a link between sporadic porphyria cutanea tarda (PCT) and human-herpesvirus-6 (HHV6) hepatitis. Sporadic PCT is a rare disease of the heme synthesis pathway. The pathogenesis has not been fully determined but iron overload and viral infections - e.g., hepatitis C virus - are thought to play an important role. We present the case of a patient suffering from myelo-monocytic leukemia. He developed symptomatic sporadic PCT concomitant with HHV6-associated subclinical hepatitis after allogeneic stem cell transplantation (SCT). Although HHV6 often reactivates after SCT and HHV6-induced hepatitis can occur in immunocompromised patients, it has not been described that HHV6 might trigger PCT. A contribution of HHV6 to the pathogenesis of sporadic PCT could have dramatic implications on our current therapeutic approach.

Elevated PIN1 expression by C/EBPalpha-p30 blocks C/EBPalpha-induced granulocytic differentiation through c-Jun in AML.

The transcription factor CCAAT enhancer-binding protein alpha (C/EBPalpha) has an important role in granulopoiesis. The tumor suppressor function of C/EBPalpha is shown by the findings that loss of expression or function of C/EBPalpha in leukemic blasts contributes to a block in myeloid cell differentiation and to leukemia. C/EBPalpha mutations are found in around 9% of acute myeloid leukemia (AML) patients. The mechanism by which the mutant form of C/EBPalpha (C/EBPalpha-p30) exerts a differentiation block is not well understood. By using a proteomic screen, we have recently reported PIN1 as a target of C/EBPalpha-p30 in AML. In the present study, we show that C/EBPalpha-p30 induces PIN1 expression. We observed elevated PIN1 expression in leukemic patient samples. Induction of C/EBPalpha-p30 results in recruitment of E2F1 in the PIN1 promoter. We show that the inhibition of PIN1 leads to myeloid differentiation in primary AML blasts with C/EBPalpha mutations. Overexpression of PIN1 in myeloid cells leads to block of granulocyte differentiation. We also show that PIN1 increases the stability of the c-Jun protein by inhibiting c-Jun ubiquitination, and c-Jun blocks granulocyte differentiation mediated by C/EBPalpha. Our data suggest that the inhibition of PIN1 could be a potential strategy of treating AML patients with C/EBPalpha mutation.

Multiple ways of C/EBPalpha inhibition in myeloid leukaemia.

Transcription factors play a crucial role in myeloid differentiation and lineage determination. Tumour suppressor protein C/EBPalpha is a key regulator of granulocytic differentiation whose functional inactivation has become a pathophysiological signature of myeloid leukaemia. In this review we describe various mechanisms such as antagonistic protein-protein interaction, mutation and posttranslational modifications of C/EBPalpha which lead to its transcriptional inhibition and render C/EBPalpha inactive in its functions.

Palifermin reduces incidence and severity of oral mucositis in allogeneic stem-cell transplant recipients.

In this multicenter study, 30 patients undergoing matched related or unrelated allogeneic stem-cell transplantation for leukemia were treated with palifermin, and retrospectively compared to a matched control group. Palifermin recipients transplanted with an unrelated donor showed a significant reduction of severity, incidence and duration of oral mucositis WHO grades 2-4. In addition, in the palifermin group the use of opioid analgesics and the duration of total parenteral nutrition decreased, whether stem cells were used from matched related or unrelated donors. No beneficial influence of palifermin on the incidence and severity of acute GVHD (aGVHD) was apparent. The incidence and duration of febrile neutropenia, documented infections, hematopoietic recovery or overall survival remained unchanged. The most common adverse effects included rash or erythema, generally mild and transient in appearance. Thus, the administration of palifermin was generally well tolerated and safe, and significantly reduced oral mucositis whereas--regardless of donor status--no effect on the incidence and severity of aGVHD was seen.

Proteomic identification of the MYST domain histone acetyltransferase TIP60 (HTATIP) as a co-activator of the myeloid transcription factor C/EBPalpha.

The transcription factor C/EBPalpha (CEBPA) is a key player in granulopoiesis and leukemogenesis. We have previously reported the interaction of C/EBPalpha with other proteins (utilizing mass spectrometry) in transcriptional regulation. In the present study, we characterized the association of the MYST domain histone acetyltransferase Tat-interactive protein (TIP) 60 (HTATIP) with C/EBPalpha. We show in pull-down and co-precipitation experiments that C/EBPalpha and HTATIP interact. A chromatin immunoprecipitation (ChIP) and a confirmatory Re-ChIP assay revealed in vivo occupancy of the C/EBPalpha and GCSF-R promoter by HTATIP. Reporter gene assays showed that HTATIP is a co-activator of C/EBPalpha. The co-activator function of HTATIP is dependent on its intact histone acetyltransferase (HAT) domain and on the C/EBPalpha DNA-binding domain. The resulting balance between histone acetylation and deacetylation at the C/EBPalpha promoter might represent an important mechanism of C/EBPalpha action. We observed a lower expression of HTATIP mRNA in undifferentiated U937 cells compared to retinoic acid-induced differentiated U937 cells, and correlated expression of CEBPA and HTATIP mRNA levels were observed in leukemia samples. These findings point to a functional synergism between C/EBPalpha and HTATIP in myeloid differentiation and suggest that HTATIP might be an important player in leukemogenesis.