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Resistance to chemotherapy is ultimately responsible for the majority of AML-related deaths, making the identification of resistance pathways a high priority. Transcriptomics approaches can be used to identify genes regulated at the level of transcription or mRNA stability but miss microRNA-mediated changes in translation, which are known to play a role in chemo-resistance. To address this, we compared miRNA profiles in paired chemo-sensitive and chemo-resistant subclones of HL60 cells and used a bioinformatics approach to predict affected pathways. From a total of 38 KEGG pathways implicated, TGF-β/activin family signaling was selected for further study. Chemo-resistant HL60 cells showed an increased TGF-β response but were not rendered chemo-sensitive by specific inhibitors. Differential pathway expression in primary AML samples was then investigated at the RNA level using publically available gene expression data in the TGCA database and by longitudinal analysis of pre- and post-resistance samples available from a limited number of patients. This confirmed differential expression and activity of the TGF-β family signaling pathway upon relapse and revealed that the expression of TGF-β and activin signaling genes at diagnosis was associated with overall survival. Our focus on a matched pair of cytarabine sensitive and resistant sublines to identify miRNAs that are associated specifically with resistance, coupled with the use of pathway analysis to rank predicted targets, has thus identified the activin/TGF-β signaling cascade as a potential target for overcoming resistance in AML.

Background: To evaluate the benefits of SARS-CoV-2 vaccination in cancer patients it is relevant to understand the adaptive immune response elicited after vaccination. Patients affected by hematologic malignancies are frequently immune-compromised and show a decreased seroconversion rate compared to other cancer patients or controls. Therefore, vaccine-induced cellular immune responses in these patients might have an important protective role and need a detailed evaluation., Methods: Certain T cell subtypes (CD4, CD8, Tfh, γδT), including cell functionality as indicated by cytokine secretion (IFN, TNF) and expression of activation markers (CD69, CD154) were assessed via multi-parameter flow cytometry in hematologic malignancy patients (N=12) and healthy controls (N=12) after a second SARS-CoV-2 vaccine dose. The PBMC of post-vaccination samples were stimulated with a spike-peptide pool (S-Peptides) of SARS-CoV-2, with CD3/CD28, with a pool of peptides from the cytomegalovirus, Epstein-Barr virus and influenza A virus (CEF-Peptides) or left unstimulated. Furthermore, the concentration of spike-specific antibodies has been analyzed in patients., Results: Our results indicate that hematologic malignancy patients developed a robust cellular immune response to SARS-CoV-2 vaccination comparable to that of healthy controls, and for certain T cell subtypes even higher. The most reactive T cells to SARS-CoV-2 spike peptides belonged to the CD4 and Tfh cell compartment, being median (IQR), 3.39 (1.41-5.92) and 2.12 (0.55-4.14) as a percentage of IFN- and TNF-producing Tfh cells in patients. In this regard, the immunomodulatory treatment of patients before the vaccination period seems important as it was strongly associated with a higher percentage of activated CD4 and Tfh cells. SARS-CoV-2- and CEF-specific T cell responses significantly correlated with each other. Compared to lymphoma patients, myeloma patients had an increased percentage of SARS-CoV-2-specific Tfh cells. T-SNE analysis revealed higher frequencies of γδT cells in patients compared to controls, especially in myeloma patients. In general, after vaccination, SARS-CoV-2-specific T cells were also detectable in patients without seroconversion., Conclusion: Hematologic malignancy patients are capable of developing a SARS-CoV-2-specific CD4 and Tfh cellular immune response after vaccination, and certain immunomodulatory therapies in the period before vaccination might increase the antigen-specific immune response. A proper response to recall antigens (e.g., CEF-Peptides) reflects immune cellular functionality and might be predictive for generating a newly induced antigen-specific immune response as is expected after SARS-CoV-2 vaccination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pfannes, Pierzchalski, Maddalon, Simion, Zouboulis, Behre, Zenclussen, Westphal, Fest and Herberth.)

Background: Monoclonal antibodies represent one option for treatment of COVID-19 early after infection. Although large clinical trials have been successfully conducted, real world data are needed to obtain a realistic assessment of the assumed effect on hospitalization rates., Methods: For this retrospective, observational study, clinical data were collected in 2021 from outpatients (402) as well as hospitalized patients (350) receiving monoclonal antibodies Bamlanivimab, Casirivimab/Imdevimab or Etesevimab/Bamlanivimab. These data were compared with data from a control group of patients not receiving antibodies because admission to the hospital was too late for this therapy., Results: Both groups showed a comparable spectrum of risk factors. Due to the late hospitalization of control patients, a higher frequency of severe symptoms, such as fever, dyspnea, syncope and lower viral load, were observed. CRP and leukocytes counts were also higher in the untreated group. Most importantly, hospitalization time was significantly shorter and the number of deaths was also lower in the treated group., Conclusions: Apparently, the application of anti-SARS-CoV-2 antibodies reduced the work load of our hospital as shown by the shorter hospitalization time and lower number of COVID-19-related deaths.

Background : Patients with steroid-refractory intestinal acute graft-versus-host disease (aGvHD) and bronchiolitis obliterans syndrome (BOS) represent a population with a high need for alternative and effective treatment options. Methods : We report real-life data from 18 patients treated with extracorporeal photopheresis (ECP). This cohort consisted of nine patients with steroid-refractory intestinal aGvHD and nine patients with BOS. Results : We document partial or complete clinical response and reduction of symptoms in half of the patients with intestinal acute GvHD and patients with BOS treated ECP. Responding patients tended to stay on treatment longer. In patients with BOS, stabilization of lung function and forced expiratory volume was observed, whereas, less abdominal pain, less diarrhea, and a reduction of systemic corticosteroids were seen in patients with intestinal acute GvHD. Conclusions : ECP might not only abrogate symptoms but also reduce mortality caused by complications from high-dose steroid treatment. Taken together, ECP offers a serious treatment avenue for patients with steroid-refractory intestinal acute GvHD and BOS.

Background: Tamoxifen-adapted MCF-7-Tam cells represent an in-vitro model for acquired tamoxifen resistance, which is still a problem in clinics. We here investigated the correlation of microRNA-, mRNA- and eukaryotic initiation factors (eIFs) expression in this model., Methods: MicroRNA- and gene expression were analyzed by nCounter and qRT-PCR technology; eIFs by Western blotting. Protein translation mode was determined using a reporter gene assay. Cells were transfected with a miR-1972-mimic., Results: miR-181b-5p,-3p and miR-455-5p were up-, miR-375, and miR-1972 down-regulated and are significant in survival analysis. About 5% of the predicted target genes were significantly altered. Pathway enrichment analysis suggested a contribution of the FoxO1 pathway. The ratio of polio-IRES driven to cap-dependent protein translation shifted towards cap-dependent initiation. Protein expression of eIF2A, -4G, -4H and -6 decreased, whereas eIF3H was higher in MCF-7-Tam. Significant correlations between tamoxifen-regulated miRNAs and eIFs were found in representative breast cancer cell lines. Transfection with a miR-1972-mimic reverses tamoxifen-induced expression for a subset of genes and increased proliferation in MCF-7, but reduced proliferation in MCF-7-Tam, especially in the presence of 4OH-tamoxifen. Migration was inhibited in MCF-7-Tam cells. Translation mode remained unaffected., Conclusions: miR-1972 contributes to the orchestration of gene-expression and physiological consequences of tamoxifen adaption.

Background: In the phase 2/3 BLAZE-1 trial, bamlanivimab and etesevimab together reduced coronavirus disease 2019 (COVID-19)-related hospitalizations and any-cause mortality in ambulatory patients. Herein, we assess the impact of bamlanivimab and etesevimab treatment on the severity and length of symptoms and health outcomes among patients at increased risk for severe COVID-19., Methods: In the phase 3 portion of BLAZE-1 (NCT04427501), symptomatic patients with increased risk for severe COVID-19 were randomized (2:1) to a single infusion of 700 mg bamlanivimab and 1400 mg etesevimab or placebo. Hospitalization events, vital signs, and symptomatology were monitored throughout the trial., Results: Overall, 769 patients were randomized to bamlanivimab and etesevimab together (n = 511) or placebo (n = 258). The time to sustained symptom resolution was significantly shorter among patients who received bamlanivimab and etesevimab compared with placebo (8 vs 10 days; P  < .01). The median time to first sustained symptom resolution of body aches and pain, chills, fatigue, feeling feverish, headache, and shortness of breath was significantly different in patients receiving bamlanivimab and etesevimab compared to placebo ( P  < .05). The proportion of patients who experienced COVID-19-related hospitalization by day 29 was significantly reduced among the bamlanivimab and etesevimab group compared with placebo (0.8% vs 5.4%; P  < .01). The mean duration of hospital stay was numerically shorter among patients who received bamlanivimab and etesevimab (7.3 vs 13.5 days; P  = .16), with fewer intensive care admissions., Conclusions: Patients receiving bamlanivimab and etesevimab together resolved their symptoms more rapidly than those receiving placebo. Bamlanivimab and etesevimab treatment was associated with reduced rates of hospitalizations and shorter hospital stays., Clinical Trials Registration: NCT04427501., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Introduction: Tamoxifen-adapted MCF-7 breast cancer cells (MCF-7-TAM-R) are a model for acquired tamoxifen resistance in oestrogen receptor-positive breast cancer. In this system, the expression of long-non-coding RNA LINC00992 is decreased. LINC00992 might therefore contribute to tamoxifen adaption and associated gene expres-sion changes. Here, we investigated whether a modulation of LINC00992 modifies gene expression, proliferation, and migration., Material and Methods: Up- and down-- regulation of LINC00992 was performed using plasmid vectors and siRNA. Gene expression was measured via nCounter® and quantitative real-time polymerase chain reaction. Database analysis was performed using GEPIA2 and cBioportal. Furthermore, we performed scratch assays, colony-forming assays, and proliferation assays with MCF-7 and MCF-7-TAM-R after up-regulation of LINC00992., Results: Up- and down-regulation of LINC00992 caused gene expression changes in 4 of the 42 tamoxifen-regulated genes tested. Especially ubiquitin D, single-minded homologue 1 (SIM1) carcinoembryonic antigen-related cell adhesion molecule 5 and the G-protein coupled oestrogen receptor 1 were affected. In tamoxifen-adapted MCF-7-TAM-R cells, LINC00992 overexpression resulted in augmented viability and proliferation and enhanced migration. Database analyses revealed that luminal breast cancers have increased expression of LINC00992 compared to Her2-type/neu- or basal type. Furthermore, higher expression of LINC00992 was associated with poor prognosis in luminal-A carcinomas., Conclusions: Changes in the expression of tamoxifen-regulated genes could be induced by manipulating LINC00992's abundance, suggesting that it is at least partially involved in the establishment of the tamoxifen-induced gene expression pattern. LINC00992 may also serve as a prognostic biomarker and may indicate the development of tamoxifen resistance., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Termedia.)

Mantle cell lymphoma (MCL) is a non-Hodgkin's lymphoma with an often aggressive course, incurable by chemotherapy. Consolidation with high-dose therapy and autologous stem cell transplantation (autoSCT) has a low transplant-related mortality but does not lead to a survival plateau. Allogeneic stem cell transplantation (alloSCT) is associated with a higher early mortality, but can cure MCL. To investigate alloSCT for therapy of MCL, we conducted two prospective trials for de novo MCL (OSHO#74) and for relapsed or refractory MCL (OSHO#60). Fifteen and 24 patients were recruited, respectively. Induction was mainly R-DHAP alternating with R-CHOP. Conditioning was either Busulfan/Cyclophosphamide or Treosulfan/Fludarabin. Either HLA-identical siblings or matched-unrelated donors with not more than one mismatch were allowed. ATG was mandatory in mismatched or unrelated transplantation. Progression-free survival (PFS) was 62% and overall survival (OS) was 68% after 16.5-year follow-up. Significant differences in PFS and OS between both trials were not observed. Patients below 56 years and patients after myeloablative conditioning had a better outcome compared to patients of the corresponding groups. Nine patients have died between day +8 and 5.9 years after SCT. Data from 7 long-term surviving patients showed an excellent Quality-of-life (QoL) after alloSCT. AlloSCT for MCL delivers excellent long-term survival data. The early mortality is higher than after autoSCT; however, the survival curves after alloSCT indicate the curative potential of this therapy. AlloSCT is a standard of care for all feasible patients with refractory or relapsed MCL and should offer to selected patients with de novo MCL and a poor risk profile. For defining the position of alloSCT in the therapeutic algorithm of MCL therapy, a randomized comparison of autoSCT and alloSCT is mandatory.

Introduction: The G protein-coupled oestrogen receptor 1 (GPER-1) is a potential prognostic marker in breast cancer. However, its role in male breast cancer (MBC) is still unknown. This study evaluates the expression of GPER-1 in MBC samples and correlates these data with clinical and pathological parameters including patients' survival., Material and Methods: For this retrospective analysis of a prospectively maintained cohort of patients with MBC, we examined 161 specimens for GPER-1 expression using immunohistochemistry. An immunoreactive score (IRS) was calculated based on staining intensity and the percentage of positive tumour cells. Then, we correlated GPER-1 IRS with clinical and pathological parameters, and overall and relapse-free survival., Results: About 40% of MBC samples were positive for GPER-1 expression (IRS ≥ 4). There was no significant correlation with clinicopathological parameters, such as hormone receptor status or grading. However, a statistical trend was observed for tumour size (≥ 2 cm, p = 0.093). Kaplan-Meier survival analysis revealed no significant correlation with relapse-free survival. However, there was a significant correlation with overall survival, but when we adjusted the log-rank p -value to compensate for the cut-off point optimization method, it rose above 0.1. Additionally, GPER-1-positive patients were older at diagnosis. When adjusted for age by multivariable Cox regression analysis, the significance of GPER-1 status for survival was further reduced., Conclusions: We found no significant prognostic value of GPER-1 in this MBC cohort as anticipated from studies on female BC. Future studies with higher sample size are needed to further verify a potential sex-specific role of GPER-1., (Copyright © 2021 Termedia.)

Allogeneic stem cell transplantation (HSCT) remains the only curative treatment for myelodysplastic syndromes (MDS) or myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients. The introduction of reduced intensity (RIC) and non-myeloablative (NMA) conditioning enabled HSCT in older or comorbid individuals representing the majority of patients. Studies comparing RIC and NMA conditioning are limited. We retrospectively analyzed 151 MDS or MDS/MPN patients older than 50 years who received NMA- or RIC-HSCT. Patients younger or older than 65 years at HSCT were analyzed separately. Patients receiving RIC-HSCT or NMA-HSCT were balanced in factors reflecting disease aggressiveness and the HCT-CI comorbidity score. The NMA conditioned patients had a higher incidence of graft rejection and chronic graft-vs-host disease. Cumulative incidence of relapse (CIR), non-relapse mortality (NRM) and overall survival (OS), did not differ significantly with regard to the conditioning regime in the whole cohort. In patients <65 years at HSCT, NMA conditioning associated with higher NRM and shorter OS by trend, while CIR was similar in both groups. In multivariable analyzes, the conditioning regimen remained a prognostic factor for NRM and OS in patients <65 years at HSCT. In MDS patients NMA and RIC conditioning result in similar disease control, but especially patients <65 years may benefit from RIC-HSCT., (© 2019 Wiley Periodicals, Inc.)

Allogeneic hematopoietic stem cell transplantation is the only curative option for a variety of diseases. Despite advances, it is associated with considerable morbidity and mortality, often involving liver complications. Liver disease can be characterized using ultrasound-based liver stiffness measurement. To assess its prognostic value, consecutive patients undergoing allogeneic hematopoietic stem cell transplantation were prospectively evaluated in a single-center study. Endpoints included liver event-free survival and all-cause mortality at 1 year. Competing risk and Cox-regression were used for analysis. We evaluated 106 patients (42 female, age 57) and observed 33 life-threatening events (14 died) including 16 liver complications at 100 days. At 1 year, 36 patients had died, 20 with disease relapse. The hazard ratios for liver-related complications at 100 days were 3.2 (95% CI: 1.8-14.6, p = 0.0022) and 4.4 (95% CI: 1.6-11.9, p = 0.0042) for elevated transient elastography (n = 11) and shear-wave velocity (n = 31), respectively. Results were analogous for all-cause mortality at 1 year. Prior stem cell therapy and elevated gamma glutamyltransferase were also associated with outcome. This demonstrates that elastography is a promising and viable tool for risk prediction and should be included in upcoming multi-center trials to establish new means of guiding treatment and prophylaxis.

Age-related somatic mutations linked to clonal hematopoiesis have been found in apparently healthy individuals and increase the risk of developing hematologic malignancies. In acute myeloid leukemia (AML) the clinical relevance of clonal hematopoiesis remains controversial and data on patients with detectable clonal hematopoiesis, consolidated with hematopoietic stem cell transplantation are limited. We analyzed samples from 113 AML patients in complete remission prior to hematopoietic stem cell transplantation for the presence of clonal hematopoiesis-associated mutations. The results were correlated with clinical and biological data. In complete remission we found 75 mutations previously linked to clonal hematopoiesis in 47 patients (41.6%). Twenty patients had ≥2 mutations linked to clonal hematopoiesis. DNMT3A, TET2, and ASXL1 were most frequently mutated. When compared to pre-treatment samples we found variable patterns of mutation persistence depending on the gene mutated. In AML patients after allogeneic hematopoietic stem cell transplantation the presence of clonal hematopoiesis-associated mutations in complete remission did not associate with inferior clinical outcome. This study demonstrates that clonal hematopoiesis is a frequent phenomenon in AML patients. Presence of clonal hematopoiesis has no negative prognostic impact in the context of an allogeneic hematopoietic stem cell transplantation and might be beneficial if certain genes are affected.

Myelodysplastic syndrome (MDS) is a highly heterogeneous clonal hematopoietic disorder. Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative treatment and is of particular interest in patients at high risk for progression to acute myeloid leukemia (AML). In MDS, CD34 + /CD38 - cells possess MDS stem cell potential, and secondary AML (sAML) clones originate from the MDS disease stage. However, the prognostic impact of the pretreatment stem cell population burden in MDS remains unknown. We retrospectively analyzed the prognostic impact of the pretreatment CD34 + /CD38 - cell burden in 124 MDS patients who received allogeneic HSCT at our institution. A high pretreatment bone marrow CD34 + /CD38 - cell burden (≥1%) was associated with worse genetic risk and a higher incidence of blast excess. Patients with a high CD34 + /CD38 - cell burden had a significantly higher cumulative incidence of MDS relapse, a higher cumulative incidence of secondary AML, and a trend for shorter overall survival after allogeneic HSCT. In multivariable analyses this prognostic impact was shown to be independent of other clinical and cytogenetic risk factors in MDS. Patients suffering MDS relapse or progression to AML also had a higher pre-treatment CD34 + /CD38 - cell burden as a continuous variable. The observed prognostic impact is likely mediated by MDS stem cells within the CD34 + /CD38 - cell population initiating MDS relapse or progression to AML. New therapeutic strategies targeting MDS stem cells might improve outcomes., (Copyright © 2019 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

High expression of the leukemia-associated gene meningioma-1 ( MN1 ) is frequently found at diagnosis of acute myeloid leukemia (AML) and associates with adverse outcomes. The presence of measurable residual disease (MRD) in complete remission (CR) indicates high risk of relapse and worse outcome in AML patients. However, the prognostic impact of MN1 expression levels as MRD marker has not been evaluated. Digital droplet polymerase chain reaction (ddPCR) is a novel technique allowing sensitive and specific absolute gene expression quantification. We retrospectively analyzed 124 AML patients who received allogeneic hematopoietic stem cell transplantation (HSCT) in CR or CR with incomplete peripheral recovery. Absolute MN1 copy numbers in peripheral blood were assessed prior to HSCT (median 7; range 0-29 days) using ddPCR. High pre-HSCT MN1 /Abelson murine leukemia viral oncogene homolog 1 gene ( ABL1 ) copy numbers associated with a higher cumulative incidence of relapse after HSCT and-in relapsing patients-shorter time to relapse. In multivariable analysis, high pre-HSCT MN1 / ABL1 copy numbers remained an independent prognosticator for relapse after HSCT. Patients with the highest pre-HSCT MN1 / ABL1 copy numbers also had the highest risk of relapse. MN1 copy number assessment also added prognostic information to nucleophosmin 1 gene ( NPM1 ) mutation- and brain and acute leukemia, cytoplasmic ( BAALC ) and Wilm's tumor gene 1 ( WT1 ) expression-based MRD evaluation. Our study demonstrates the feasibility of the novel ddPCR technique for MN1 / ABL1 copy number assessment as a marker for MRD. Evaluation of MN1 / ABL1 copy numbers allows the identification of patients at high risk of relapse, independently of other diagnostic risk factors and MRD markers., (Copyright © 2018 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Invasive fungal diseases (IFD) are a primary cause of morbidity and mortality in patients with haematological malignancies. These infections are mostly life-threatening and an early diagnosis and initiation of appropriate antifungal therapy are essential for the clinical outcome. Most commonly, Aspergillus and Candida species are involved. However, other Non-Aspergillus moulds are increasingly identified in case of documented IFD. For definite diagnosis of IFD, a combination of diagnostic tools have to be applied, including conventional mycological culture and non-conventional microbiological tests such as antibody/antigen and molecular tests, as well as histopathology and radiology. Although varying widely in cancer patients, the risk of invasive fungal infection is highest in those with allogeneic stem cell transplantation and those with acute leukaemia and markedly lower in patients with solid cancer. Since the last edition of Diagnosis of Invasive Fungal Diseases recommendations of the German Society for Hematology and Oncology in 2012, integrated care pathways have been proposed for the management and therapy of IFDs with either a diagnostic driven strategy as opposed to a clinical or empirical driven strategy. This update discusses the impact of this additional evidence and effective revisions., (© 2018 Blackwell Verlag GmbH.)

Despite the constant development of innovative therapeutic options for hematological malignancies, the gold-standard therapy regimen for curative treatment often includes allogeneic hematopoietic stem cell transplantation (HSCT). The graft-vs.-leukemia effect (GVL) is one of the main therapeutic goals that arises from HSCT. On the other hand, graft-vs.-host disease (GVHD) is still one of the main and most serious complications following allogeneic HSCT. In acute myeloid leukemia (AML), HSCT together with high-dose chemotherapy is used as a treatment option. An aggressive progression of the disease, a decreased response to treatment, and a poor prognosis are connected to internal tandem duplication (ITD) mutations in the Fms like tyrosine kinase 3 (FLT3) gene, which affects around 30% of AML patients. In this study, C3H/HeN mice received an allogeneic graft together with 32D-FLT3 ITD AML cells to induce acute GVHD and GVL. It was examined if pre-incubation of the graft with the anti-human cluster of differentiation (CD) 4 antibody MAX.16H5 IgG 1 prevented the development of GVHD and whether the graft function was impaired. Animals receiving grafts pre-incubated with the antibody together with FLT3 ITD AML cells survived significantly longer than mice receiving untreated grafts. The observed prolonged survival due to MAX.16H5 incubation of immune cell grafts prior to transplantation may allow an extended application of additional targeted strategies in the treatment of AML.

Allogeneic hematopoietic stem cell transplantation is an established consolidation therapy for patients with acute myeloid leukemia. However, relapse after transplantation remains a major clinical problem resulting in poor prognosis. Thus, detection of measurable ("minimal") residual disease to identify patients at high risk of relapse is essential. A feasible method to determine measurable residual disease may be digital droplet PCR (ddPCR) that allows absolute quantification with high sensitivity and specificity without the necessity of standard curves. Using ddPCR, we analyzed pre-transplant peripheral blood and bone marrow of 51 NPM1-mutated acute myeloid leukemia patients transplanted in complete remission or complete remission with incomplete recovery. Mutated NPM1 measurable residual disease-positive patients had higher cumulative incidence of relapse (P < 0.001) and shorter overall survival (P = 0.014). Restricting the analyses to patients receiving non-myeloablative conditioning, mutated NPM1 measurable residual disease positivity is associated with higher cumulative incidence of relapse (P < 0.001) and shorter overall survival (P = 0.006). Positive mutated NPM1 measurable residual disease status determined by ddPCR before allogeneic stem cell transplantation is associated with worse prognosis independent of other known prognostic markers-also for those receiving non-myeloablative conditioning. In the future, mutated NPM1 measurable residual disease status determined by ddPCR might guide treatment and improve patients' outcomes.

Hematopoiesis, the formation of blood cells from hematopoietic stem cells (HSC), is a highly regulated process. Since the discovery of microRNAs (miRNAs), several studies have shown their significant role in the regulation of the hematopoietic system. Impaired expression of miRNAs leads to disrupted cellular pathways and in particular causes loss of hematopoietic ability. Here, we report a previously unrecognized function of miR-143 in granulopoiesis. Hematopoietic cells undergoing granulocytic differentiation exhibited increased miR-143 expression. Overexpression or ablation of miR-143 expression resulted in accelerated granulocytic differentiation or block of differentiation, respectively. The absence of miR-143 in mice resulted in a reduced number of mature granulocytes in blood and bone marrow. Additionally, we observed an association of high miR-143 expression levels with a higher probability of survival in two different cohorts of patients with acute myeloid leukemia (AML). Overexpression of miR-143 in AML cells impaired cell growth, partially induced differentiation, and caused apoptosis. Argonaute2-RNA-Immunoprecipitation assay revealed ERK5, a member of the MAPK-family, as a target of miR-143 in myeloid cells. Further, we observed an inverse correlation of miR-143 and ERK5 in primary AML patient samples, and in CD34 + HSPCs undergoing granulocytic differentiation and we confirmed functional relevance of ERK5 in myeloid cells. In conclusion, our data describe miR-143 as a relevant factor in granulocyte differentiation, whose expression may be useful as a prognostic and therapeutic factor in AML therapy.

Immunocompromised patients are at high risk of invasive fungal infections (IFI), in particular those with haematological malignancies undergoing remission-induction chemotherapy for acute myeloid leukaemia (AML) or myelodysplastic syndrome (MDS) and recipients of allogeneic haematopoietic stem cell transplants (HSCT). Despite the development of new treatment options in the past decades, IFI remains a concern due to substantial morbidity and mortality in these patient populations. In addition, the increasing use of new immune modulating drugs in cancer therapy has opened an entirely new spectrum of at risk periods. Since the last edition of antifungal prophylaxis recommendations of the German Society for Haematology and Medical Oncology in 2014, seven clinical trials regarding antifungal prophylaxis in patients with haematological malignancies have been published, comprising 1227 patients. This update assesses the impact of this additional evidence and effective revisions. Our key recommendations are the following: prophylaxis should be performed with posaconazole delayed release tablets during remission induction chemotherapy for AML and MDS (AI). Posaconazole iv can be used when the oral route is contraindicated or not feasible. Intravenous liposomal amphotericin B did not significantly decrease IFI rates in acute lymphoblastic leukaemia (ALL) patients during induction chemotherapy, and there is poor evidence to recommend it for prophylaxis in these patients (CI). Despite substantial risk of IFI, we cannot provide a stronger recommendation for these patients. There is poor evidence regarding voriconazole prophylaxis in patients with neutropenia (CII). Therapeutic drug monitoring TDM should be performed within 2 to 5 days of initiating voriconazole prophylaxis and should be repeated in case of suspicious adverse events or of dose changes of interacting drugs (BIItu). General TDM during posaconazole prophylaxis is not recommended (CIItu), but may be helpful in cases of clinical failure such as breakthrough IFI for verification of compliance or absorption.

Active BCR related ( ABR ) gene deactivates ras-related C3 botulinum toxin substrate 1 (RAC1), which plays an essential role in regulating normal hematopoiesis and in leukemia. BCR gene, closely related to ABR, acts as a tumor suppressor in chronic myeloid leukemia and has overlapping functions with ABR . Evidence for a putative tumor suppressor role of ABR has been shown in several solid tumors, in which deletion of ABR is present. Our results show downregulation of ABR in AML. A block of ABR prevents myeloid differentiation and leads to repression of the myeloid transcription factor C/EBPα, a major regulator of myeloid differentiation and functionally impaired in leukemia. Conversely, stable overexpression of ABR enhances myeloid differentiation. Inactivation of the known ABR target RAC1 by treatment with the RAC1 inhibitor NSC23766 resulted in an increased expression of C/EBPα in primary AML samples and in AML cell lines U937 and MV4;11. Finally, AML patients with high ABR expression at diagnosis showed a significant longer overall survival and patients who respond to azacitidine therapy showed a significant higher ABR expression. This is the first report showing that ABR expression plays a critical role in both myelopoiesis and AML. Our data indicate the tumor suppressor potential of ABR and underline its potential role in leukemia therapeutic strategies., Competing Interests: CONFLICTS OF INTEREST None.

High BAALC expression levels at acute myeloid leukemia diagnosis have been linked to adverse outcomes. Recent data indicate that high BAALC expression levels may also be used as marker for residual disease following acute myeloid leukemia treatment. Allogeneic hematopoietic stem cell transplantation (HSCT) offers a curative treatment for acute myeloid leukemia patients. However, disease recurrence remains a major clinical challenge and identification of high-risk patients prior to HSCT is crucial to improve outcomes. We performed absolute quantification of BAALC copy numbers in peripheral blood prior (median 7 days) to HSCT in complete remission (CR) or CR with incomplete peripheral recovery in 82 acute myeloid leukemia patients using digital droplet PCR (ddPCR) technology. An optimal cut-off of 0.14 BAALC / ABL1 copy numbers was determined and applied to define patients with high or low BAALC / ABL1 copy numbers. High pre-HSCT BAALC / ABL1 copy numbers significantly associated with higher cumulative incidence of relapse and shorter overall survival in univariable and multivariable models. Patients with high pre-HSCT BAALC / ABL1 copy numbers were more likely to experience relapse within 100 days after HSCT. Evaluation of pre-HSCT BAALC / ABL1 copy numbers in peripheral blood by ddPCR represents a feasible and rapid way to identify acute myeloid leukemia patients at high risk of early relapse after HSCT. The prognostic impact was also observed independently of other known clinical, genetic, and molecular prognosticators. In the future, prospective studies should evaluate whether acute myeloid leukemia patients with high pre-HSCT BAALC / ABL1 copy numbers benefit from additional treatment before or early intervention after HSCT., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Transcription factor C/EBPα is a master regulator of myelopoiesis and its inactivation is associated with acute myeloid leukemia. Deregulation of C/EBPα by microRNAs during granulopoiesis or acute myeloid leukemia development has not been studied. Here we show that oncogenic miR-182 is a strong regulator of C/EBPα. Moreover, we identify a regulatory loop between C/EBPα and miR-182. While C/EBPα blocks miR-182 expression by direct promoter binding during myeloid differentiation, enforced expression of miR-182 reduces C/EBPα protein level and impairs granulopoiesis in vitro and in vivo. In addition, miR-182 expression is highly elevated particularly in acute myeloid leukemia patients with C-terminal CEBPA mutations, thereby depicting a mechanism by which C/EBPα blocks miR-182 expression. Furthermore, we present miR-182 expression as a prognostic marker in cytogenetically high-risk acute myeloid leukemia patients. Our data demonstrate the importance of a controlled balance between C/EBPα and miR-182 for the maintenance of healthy granulopoiesis.C/EBPα is a critical transcription factor involved in myelopoiesis and its inactivation is associated with acute myeloid leukemia (AML). Here the authors show a negative feedback loop between C/EBPα and miR-182 and identify this miRNA as a marker of high-risk AML.

In acute myeloid leukemia (AML), leukemia-initiating cells exist within the CD34+/CD38- cell compartment. They are assumed to be more resistant to chemotherapy, enriched in minimal residual disease cell populations, and responsible for relapse. Here we evaluated clinical and biological associations and the prognostic impact of a high diagnostic CD34+/CD38- cell burden in 169 AML patients receiving an allogeneic stem cell transplantation in complete remission. Here, the therapeutic approach is mainly based on immunological graft-versus-leukemia effects. Percentage of bone marrow CD34+/CD38- cell burden at diagnosis was measured using flow cytometry and was highly variable (median 0.5%, range 0%-89% of all mononuclear cells). A high CD34+/CD38- cell burden at diagnosis associated with worse genetic risk and secondary AML. Patients with a high CD34+/CD38- cell burden had shorter relapse-free and overall survival which may be mediated by residual leukemia-initiating cells in the CD34+/CD38- cell population, escaping the graft-versus-leukemia effect after allogeneic transplantation. Evaluating the CD34+/CD38- cell burden at diagnosis may help to identify patients at high risk of relapse after allogeneic transplantation. Further studies to understand leukemia-initiating cell biology and develop targeting therapies to improve outcomes of AML patients are needed., (© 2017 Wiley Periodicals, Inc.)

Development of hematopoietic populations through the process of differentiation is critical for proper hematopoiesis. The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) is a master regulator of myeloid differentiation, and the identification of C/EBPα target genes is key to understand this process. Here we identified the Ecotropic Viral Integration Site 2B (EVI2B) gene as a direct target of C/EBPα. We showed that the product of the gene, the transmembrane glycoprotein EVI2B (CD361), is abundantly expressed on the surface of primary hematopoietic cells, the highest levels of expression being reached in mature granulocytes. Using shRNA-mediated downregulation of EVI2B in human and murine cell lines and in primary hematopoietic stem and progenitor cells, we demonstrated impaired myeloid lineage development and altered progenitor functions in EVI2B-silenced cells. We showed that the compromised progenitor functionality in Evi2b-depleted cells can be in part explained by deregulation of cell proliferation and apoptosis. In addition, we generated an Evi2b knockout murine model and demonstrated altered properties of hematopoietic progenitors, as well as impaired G-CSF dependent myeloid colony formation in the knockout cells. Remarkably, we found that EVI2B is significantly downregulated in human acute myeloid leukemia samples characterized by defects in CEBPA. Altogether, our data demonstrate that EVI2B is a downstream target of C/EBPα, which regulates myeloid differentiation and functionality of hematopoietic progenitors.

Competing Interests: The authors have declared that no competing interests exist.

Introduction: Hematopoietic stem cell transplantation (HCT) is considered a standard treatment for high-risk acute myeloid leukemia (AML) in first or second complete remission (CR). Unfortunately, not all patients achieve complete remission prior to HCT. We sought to establish predictive factors for survival after HCT for refractory AML after FLAMSA-RIC., Patients and Methods: We analyzed the outcome of 44 consecutive patients aged between 21 and 65 years transplanted at the University Hospitals of Jena and Leipzig for refractory AML between 2006 and January 2013. Conditioning for HCT was performed with chemotherapy consisting of fludarabine, cytarabine, and amsacrine followed by total body irradiation or busulfan combined with cyclophosphamide. Antithymocyte globulin was given when transplanting from unrelated donors (FLAMSA-RIC)., Results: Estimated overall survival (OS) and event-free survival (EFS) at 3 years after a median follow-up of 34 (range 6-71) months were 15 and 12 %, respectively. Causes of death were relapse in 66 %, infection in 11 %, and graft-versus-host disease (GvHD) in 7 % of all patients. Twenty-five from 42 evaluable patients (60 %) achieved CR 4 weeks after HCT, while eight patients had partial remission (PR), and nine patients had stable disease (SD). Another six patients with PR and SD achieved CR (overall CR rate 74 %) from 4 weeks to day 90 after HCT following reduction in immunosuppression. The strongest favorable factors in univariate analysis for OS, EFS, and RI were ≥98 % total donor chimerism 2-4 weeks after HCT and <3 lines of pretreatment prior to HCT. In addition, better OS was detected in patients with <20 % bone marrow blasts alone (32 vs. 5 % at 3 years) and in combination with <3 lines of pretreatment (38 vs. 4 % at 3 years). Only a trend for better EFS and lower RI was observed in patients with limited chronic GvHD. In addition, a lower RI was seen in patients with <5 % blasts 4 weeks after HCT. Multivariate analysis revealed that ≥98 % donor chimerism 2-4 weeks after HCT for OS, EFS, and RI and <3 lines of pretreatment for OS and EFS are the strongest predictors for better outcome., Conclusion: FLAMSA-RIC shows long-term survival in refractory AML patients. Factors for favorable outcome are <20 % bone marrow blasts prior to HCT, <3 lines of pretreatment and complete donor chimerism after HCT.

In acute promyelocytic leukemia (APL), all-trans retinoic acid (ATRA) treatment induces granulocytic maturation and complete remission of leukemia. microRNAs are known to be critical players in the formation of the leukemic phenotype. In this study, we report downregulation of the miR-181a/b gene cluster in APL blasts and NB4 leukemia cells upon ATRA treatment as a key event in the drug response. We found that miR-181a/b expression was activated by the PML/RARα oncogene in cells and transgenic knock-in mice, an observation confirmed and extended by evidence of enhanced expression of miR-181a/b in APL patient specimens. RNA interference (RNAi)-mediated attenuation of miR-181a/b expression in NB4 cells was sufficient to reduce colony-forming capacity, proliferation, and survival. Mechanistic investigations revealed that miR-181a/b targets the ATRA-regulated tumor suppressor gene RASSF1A by direct binding to its 3'-untranslated region. Enforced expression of miR-181a/b or RNAi-mediated attenuation of RASSF1A inhibited ATRA-induced granulocytic differentiation via regulation of the cell-cycle regulator cyclin D1. Conversely, RASSF1A overexpression enhanced apoptosis. Finally, RASSF1A levels were reduced in PML/RARα knock-in mice and APL patient samples. Taken together, our results define miR-181a and miR-181b as oncomiRs in PML/RARα-associated APL, and they reveal RASSF1A as a pivotal element in the granulocytic differentiation program induced by ATRA in APL., (©2015 American Association for Cancer Research.)

Introduction: Bortezomib is a proteasome inhibitor that has shown important clinical efficacy either as a single agent or in combination in patients with multiple myeloma (MM). In the present protocol, bortezomib was combined with bendamustine and prednisone, in order to assess the efficacy and safety of this combination therapy in patients with newly diagnosed/untreated MM., Methods: Between June 2006 and October 2013, 49 patients with newly diagnosed/untreated MM were treated with bendamustine 60 mg/m(2) on days 1 and 2, bortezomib 1.3 mg/m(2) on days 1, 4, 8 and 11, and prednisone 100 mg on days 1, 2, 4, 8 and 11 bendamustine, prednisone and bortezomib (BPV) once every 21 days. Patients were divided into three groups: group A (n = 19) consisted of patients with normal renal function or mild dysfunction (eGFR ≥ 60 ml/min), group B (n = 15) patients with moderate or severe renal dysfunction (eGFR 15-59 ml/min) and group C (n = 15) patients with renal failure/dialysis (eGFR <15 ml/min)., Results: A median number of two (range 1-5) BPV treatment cycles were given to the patients. The majority of the patients (n = 40, 82 %) responded after at least one cycle of BPV therapy with five stringent complete responses (CRs), nine near complete responses, 12 very good partial responses and 14 partial responses. Five patients had MR, three stable and one progressive disease. After a median observation time of 13 months, progression-free survival (PFS) and overall survival (OS) at 12 months were 92 and 94 %, respectively, for patients with normal renal function or mild renal dysfunction (group A) and 83 and 93 %, respectively, for patients with moderate or severe renal dysfunction (group B). Outcome for these patients was slightly better but not statistically significantly better than that for patients with renal failure/dialysis (group C), who had a PFS, and OS of 66 % (p = 0.08) and 73 % (p = 0.05), respectively. These results indicate that this BPV combination is feasible, effective and well tolerated in patients with newly diagnosed MM and normal or impaired renal function.

This study was conducted in order to evaluate allogeneic stem cell transplantation (alloSCT) as consolidation for patients with mantle cell lymphoma (MCL). Patients with MCL were included into two prospective trials OSHO #060 (refractory/relapsed) and #074 (de novo). Induction was rituximab and chemotherapy. Responding patients proceeded to alloSCT. Minimal residual disease was monitored by quantitative RT-PCR detecting either t(11;14) or clonospecific CDR-III regions. In case of circulating lymphoma cells, immunomodulation (cyclosporine A withdrawal, rituximab, donor lymphocyte infusion) was initiated. Thirty-three of 39 patients underwent alloSCT after myeloablative (n = 7) or toxicity-reduced (n = 26) conditioning. Leukocytes engrafted at day +16 (median, range 0-101) and platelets at day +14 (0-142). Acute graft-versus-host disease stages I-II occurred in 42 % and stages III-IV in 15 %. Five patients have relapsed after SCT. The overall mortality after SCT was 24 % (n = 8). Median follow-up after SCT was 2.8 years (range 0.0-10.9). Five-year progression-free survival was 67 %, and overall survival 73 % after SCT. The results were comparable for primary MCL and refractory/relapsed disease as well as for related vs. unrelated SCT. Younger patients had a significantly better outcome than the elderly. AlloSCT is a feasible and promising consolidation therapy for relapsed and refractory disease and an attractive option for young patients with de novo MCL of high risk.

Abstract Reconstitution, engraftment kinetics and tumor cell clearance were analyzed after reduced intensity conditioning hematopoietic cell transplant (RIC-HCT) in patients with chronic lymphocytic leukemia (CLL). Patients were transplanted from unrelated (n = 40) or related (n = 10) donors after fludarabine and 2 Gy total body irradiation followed by cyclosporine and mycophenolate mofetil. The vast majority of patients (96%) engrafted with absolute neutrophil count (ANC) > 0.5 × 10(9)/L at day + 22. CLL cells decreased (median 2%, range 0-69%) within 28 days, but disappeared by day + 180 after HCT. Donor T-cell chimerism increased to > 95% at day 56 and donor B-cell chimerism to 94% at day + 360. Overall survival was 51 ± 8%, incidence of progression 37 ± 7% and non-relapse related mortality (NRM) 30 ± 7% at 4 years. The most common causes of NRM were graft-versus-host disease (GvHD) (14%) and sepsis (6%). Disease status at HCT was significantly associated with early B-cell reconstitution (p = 0.04) and with increased risk of relapse/progression in univariate and multivariate analysis (p = 0.022). Tumor cells were undetectable by day + 180, although B-cell reconstitution did not occur until 1.5 years after RIC-HCT. The best predictors for progression-free survival (PFS) and overall survival (OS) were complete response (CR) or first partial response (PR1) and the absence of bulky disease at transplant, respectively.

This is the first report showing that an epitope-specific ex vivo modulation of an allogeneic hematopoietic stem cell graft by the anti-human CD4 antibody MAX.16H5 IgG1 simultaneously facilitates the anti-tumor capacity of the graft (Graft-versus-leukemia effect, GvL) and the long-term suppression of the deleterious side effect Graft-versus-host-disease (GvHD). To distinguish and consolidate GvL from GvHD, the anti-human CD4 antibody MAX16.H5 IgG1 was tested in murine GvHD and tumor models. The survival rate was significantly increased in recipients receiving a MAX.16H5 IgG1 short-term (2 h) pre-incubated graft even when tumor cells were co-transplanted or when recipient mice were treated by MAX.16H5 IgG1 before transplantation. After engraftment, regulatory T-cells are generated only supporting the GvL effect. It was also possible to transfer the immune tolerance from GvHD-free recipient chimeras into third party recipient mice without the need of reapplication of MAX.16H5 IgG1 anti-human CD4 antibodies. These findings are also benefical for patients with leukemia when no matched related or unrelated donor is available and provides a safer allogeneic HSCT, which is more effective against leukemia. It also facilitates allogeneic (stem) cell transplantations for other indications (e.g., autoimmune-disorders).

The basic leucine zipper transcription factor CCAAT/enhancer binding protein alpha (CEBPA) codes for a critical regulator during neutrophil differentiation. Aberrant expression or function of this protein contributes to the development of acute myeloid leukemia (AML). In this study, we identified two novel unrelated CEBPA target genes, the glycolytic enzyme hexokinase 3 (HK3) and the krüppel-like factor 5 (KLF5) transcription factor, by comparing gene profiles in two cohorts of CEBPA wild-type and mutant AML patients. In addition, we found CEBPA-dependent activation of HK3 and KLF5 transcription during all-trans retinoic acid (ATRA) mediated neutrophil differentiation of acute promyelocytic leukemia (APL) cells. Moreover, we observed direct regulation of HK3 by CEBPA, whereas our data suggest an indirect regulation of KLF5 by this transcription factor. Altogether, our data provide an explanation for low HK3 and KLF5 expression in particular AML subtype and establish these genes as novel CEBPA targets during neutrophil differentiation.

DAPK2 is a proapoptotic protein that is mostly expressed in the hematopoietic tissue. A detailed DAPK2 expression analysis in two large AML patient cohorts revealed particularly low DAPK2 mRNA levels in APL. DAPK2 levels were restored in APL patients undergoing ATRA therapy. PML-RARA is the predominant lesion in APL causing transcriptional repression of genes important for neutrophil differentiation. We found binding of PML-RARA and PU.1, a myeloid master regulator, to RARA and PU.1 binding sites in the DAPK2 promoter. Ectopic expression of PML-RARA in non-APL, as well as knocking down PU.1 in APL cells, resulted in a significant reduction of DAPK2 expression. Restoring DAPK2 expression in PU.1 knockdown APL cells partially rescued neutrophil differentiation, thereby identifying DAPK2 as a relevant PU.1 downstream effector. Moreover, low DAPK2 expression is also associated with C/EBPα-mutated AML patients, and we found C/EBPα-dependent regulation of DAPK2 during APL differentiation. In conclusion, we identified first inhibitory mechanisms responsible for the low DAPK2 expression in particular AML subtypes, and the regulation of DAPK2 by two myeloid transcription factors underlines its importance in neutrophil development.

Tight control between activation and attenuation of granulocyte colony stimulating factor receptor (G-CSFR) signaling is essential to regulate survival, proliferation and differentiation of myeloid progenitor cells. Previous studies demonstrated negative regulation of G-CSFR through endosomal-lysosomal routing and ubiquitin-proteasome mediated degradation. However, very few E3 ubiquitin ligases are known to target G-CSFR for ubiquitin-proteasome pathway. Here we identified F-box and WD repeat domain-containing 7 (Fbw7), a substrate recognizing component of Skp-Cullin-F box (SCF) E3 ubiquitin Ligase physically associates with G-CSFR and promotes its ubiquitin-mediated proteasomal degradation. Our data shows that Fbw7 also interacts with and degrades G-CSFR-T718 (a truncated mutant of G-CSFR found in severe congenital neutropenia/acute myeloid leukemia (SCN/AML patients)) though at a quite slower rate compared to G-CSFR. We further show that glycogen synthase kinase 3 beta (GSK3β), like Fbw7 also targets G-CSFR and G-CSFR-T718 for degradation; however, Fbw7 and GSK3β are interdependent in targeting G-CSFR/G-CSFR-T718 for degradation because they are unable to degrade G-CSFR individually when either of them is knocked down. We further show that Fbw7 mediated downregulation of G-CSFR inhibits signal transducer and activator of transcription 3 (STAT3) phosphorylation which is required for G-CSF dependent granulocytic differentiation. In addition, our data also shows that inhibition of Fbw7 restores G-CSFR signaling leading to enhanced STAT3 activity resulting in massive granulocytic differentiation. These data indicate that Fbw7 together with GSK3β negatively regulates G-CSFR expression and its downstream signaling., (© 2013.)

The transcription factor CCAAT enhancer binding protein α (C/EBPα) is a master regulator in granulopoiesis and is frequently disrupted in acute myeloid leukemia (AML). We have previously shown that C/EBPα exerts its effects by regulating microRNAs (miRs) such as miR-223 and miR-34a. Here, we confirm miR-30c as a novel important target of C/EBPα during granulopoiesis. Thus, wild-type C/EBPα-p42 directly upregulates miR-30c expression, whereas C/EBPα-p30, found in AML, does not. miR-30c is downregulated in AML, especially in normal karyotype AML patients with CEBPA mutations. An induced C/EBPα knockout in mice leads to a significant downregulation of miR-30c expression in bone marrow cells. We identified NOTCH1 as a direct target of miR-30c. Finally, a block of miR-30c prevents C/EBPα-induced downregulation of Notch1 protein and leads to a reduced CD11b expression in myeloid differentiation. Our study presents the first evidence that C/EBPα, miR-30c, and Notch1 together play a critical role in granulocytic differentiation and AML, and particularly in AML with CEBPA mutations. These data reveal the importance of deregulated miRNA expression in leukemia and may provide novel biomarkers and therapeutic targets in AML.

Nuclear receptor coregulators play an important role in the transcriptional regulation of nuclear receptors. In the present study, we aimed to identify estrogen receptor α (ERα) interacting proteins in Tamoxifen treated MCF7 cells. Using in vitro GST-pull down assay with ERα ligand-binding domain (ERα-LBD) and MS-based proteomics approach we identified Profilin1 as a novel ERα interacting protein. Profilin1 contains I/LXX/L/H/I amino acid signature motif required for corepressor interaction with ERα. We show that these two proteins physically interact with each other both in vitro as well as in vivo by GST-pull down and coimmunoprecipitation, respectively. We further show that these two proteins also colocalize together in the nucleus. Previous studies have reported reduced expression of Profilin1 in breast cancer; and here we found that Tamoxifen increases Profilin1 expression in MCF7 cells. Our data demonstrate that over expression of Profilin1 inhibits ERα-mediated transcriptional activation as well as its downstream target genes in ERα positive breast cancer cells MCF7. In addition, Profilin1 overexpression in MCF7 cells leads to inhibition of cell proliferation that apparently is due to enhanced apoptosis. In nutshell, these data indicate that MS-based proteomics approach identifies a novel ERα interacting protein Profilin1 that serves as a putative corepressor of ERα functions., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CCAAT/Enhancer Binding Protein Alpha (C/EBPα) is a key transcription factor involved in the adipocyte differentiation. Here for the first time we demonstrate that E6AP, an E3 ubiquitin ligase inhibits adipocyte differentiation in 3T3-L1 cells as revealed by reduced lipid staining with oil red. Knock down of E6AP in mouse 3T3L1 preadipocytes is sufficient to convert them to adipocytes independent of external hormonal induction. C/EBPα protein level is drastically increased in E6AP deficient 3T3L1 preadipocytes while inverse is observed when wild type E6AP is over expressed. We show that transient transfection of wild type E6AP downregulates C/EBPα protein expression in a dose dependent manner while catalytically inactive E6AP-C843A rather stabilizes it. In addition, wild type E6AP inhibits expression of proadipogenic genes while E6AP-C843A enhances them. More importantly, overexpression of E6AP-C843A in mesenchymal progenitor cells promotes accumulation of lipid droplets while there is drastically reduced lipid droplet formation when E6AP is over expressed. Taken together, our finding suggests that E6AP may negatively control adipogenesis by inhibiting C/EBPα expression by targeting it to ubiquitin-proteasome pathway for degradation.

Adipogenesis is the differentiation of preadipocytes to adipocytes which is marked by the accumulation of lipid droplets. Adipogenic differentiation of 3T3-L1 cells is achieved by exposing the cells to Insulin, Dexamethasone and IBMX for 5-7 days. Thiazolidinedione drugs, like rosiglitazone are potent insulin sensitizing agents and have been shown to enhance lipid droplet formation in 3T3-L1 cells, a model cell line for preadipocyte differentiation. Guggulsterone is a natural drug extracted from the gum resin of tree Commiphora mukul. Guggulsterone has been shown to inhibit adipogenesis and induce apoptosis in 3T3-L1 cells. In this study we treated the 3T3-L1 preadipocytes with rosiglitazone and guggulsterone and assessed the protein expression profile using 2D gel electrophoresis-based proteomics to find out differential target proteins of these drugs. The proteins that were identified upon rosiglitazone treatment generally regulate cell proliferation and/or exhibit anti-inflammatory effect which strengthens its differentiation-inducing property. Guggulsterone treatment resulted in the identification of the apoptosis-inducing proteins to be up regulated which rightly is in agreement with the apoptosis-inducing property of guggulsterone in 3T3-L1 cells. Some of the proteins identified in our proteomic screen such as Galectin1, AnnexinA2 & TCTP were further confirmed by Real Time qPCR. Thus, the present study provides a better outlook of proteins being differentially regulated/expressed upon treatment with rosiglitazone and guggulsterone. The detailed study of the differentially expressed proteins identified in this proteomic screen may further provide the better molecular insight into the mode of action of these anti-diabetic drugs rosiglitazone and guggulsterone.

Background: We report a prospective multicenter phase II study of haploidentical hematopoietic stem cell transplantation using CD3/CD19-depleted grafts after reduced intensity conditioning with fludarabine, thiotepa, melphalan and OKT-3., Design and Methods: Sixty-one adults with a median age of 46 years (range 19-65 years) have been enrolled. Diagnoses were acute myeloid leukemia (n=38), acute lymphoblastic leukemia (n=8), non-Hodgkin's lymphoma (n=6), myeloma (n=4), chronic myeloid leukemia (n=3), chronic lymphatic leukemia (n=1) and myelodysplastic syndrome (n=1). Patients were considered high risk because of refractory disease (n=18), cytogenetics (n=6), complete remission (≥ 2) (n=9), chemosensitive relapse in partial remission (n=4) or relapse after prior hematopoietic stem cell transplantation (n=15 allogeneic, n=8 autologous, n=1 both). At haploidentical hematopoietic stem cell transplantation, 30 patients were in complete remission and 31 in partial remission. Grafts contained a median of 7.0 × 10(6) (range 3.2-22) CD34(+) cells/kg, 4.2 × 10(4) (range 0.6-44) CD3(+) T cells/kg and 2.7 × 10(7) (range 0.00-37.3) CD56(+) cells/kg., Results: Engraftment was rapid with a median of 12 days to granulocytes more than 0.5 × 10(9)/L (range 9-50 days) and 11 days to platelets more than 20 × 10(9) (range 7-38 days). Incidence of grade IIIV acute graft-versus-host-disease and chronic graft-versus-host-disease was 46% and 18%, respectively. Non-relapse mortality on Day 100 was 23% and 42% at two years. Cumulative incidence of relapse/progression at two years was 31%. Kaplan-Meier estimated 1-year and 2-year overall survival with median follow up of 869 days (range 181-1932) is 41% and 28%, respectively., Conclusions: This regimen allows successful haploidentical hematopoietic stem cell transplantation with reduced intensity conditioning in high-risk patients lacking a suitable donor. (clinicaltrials.gov identifier:NCT00202917).

Tamoxifen (Tam) is most widely used selective estrogen receptor modulator (SERM) for treatment of hormone-responsive breast cancer. Despite being regularly used in clinical therapy for breast cancer since 1971, the mechanism of Tam action remains largely unclear. In order to gain insights into Tam-mediated antibreast cancer actions, we applied 2DE and MS based proteomics approach to identify target proteins of Tam. We identified E6-associated protein, i.e. E6AP (UBE3A) among others to be regulated by Tam that otherwise is upregulated in breast tumors. We confirmed our 2DE finding by immunoblotting and further show that Tam leads to inhibition of E6AP expression presumably by promoting its autoubiquitination, which is coupled with nuclear export and subsequent proteasome-mediated degradation. Furthermore, we show that Tam- and siE6AP-mediated inhibition of E6AP leads to enhanced G0-G1 growth arrest and apoptosis, which is also evident from significant upregulation of cytochrome-c, Bax, p21, and PARP cleavage. Taken together, our data suggest that, Tam-targeted E6AP inhibition is in fact required for Tam-mediated antibreast cancer actions. Thus, E6AP may be a therapeutic target in breast cancer., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)