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Summary: Growth factor independence 1 (GFI1) is a transcriptional repressor protein that plays an essential role in the differentiation of myeloid and lymphoid progenitors. We and other groups have shown that GFI1 has a dose‐dependent role in the initiation, progression, and prognosis of acute myeloid leukaemia (AML) patients by inducing epigenetic changes. We now demonstrate a novel role for dose‐dependent GFI1 expression in regulating metabolism in haematopoietic progenitor and leukaemic cells. Using in‐vitro and ex‐vivo murine models of MLL::AF9‐induced human AML and extra‐cellular flux assays, we now demonstrate that a lower GFI1 expression enhances oxidative phosphorylation rate via upregulation of the FOXO1‐ MYC axis. Our findings underscore the significance of therapeutic exploitation in GFI1‐low‐expressing leukaemia cells by targeting oxidative phosphorylation and glutamine metabolism. [ABSTRACT FROM AUTHOR]

Simple Summary: Multiple myeloma (MM) is characterized by the clonal accumulation of abnormal plasma cells in the bone marrow. Although current treatments have improved the survival rates of patients with MM, MM remains incurable for most patients due to refractory disease and relapse. Thus, new therapeutic strategies are needed. Constitutive activation of the PI3K/AKT/mTOR signaling pathway has been identified in MM, leading to uncontrolled tumor growth and survival. In this study, we revealed that an inhibitor targeting mTOR with temsirolimus exhibited different anti-proliferation activity across our MM cell lines, regardless of their genetic features. Combination treatment of the mTOR inhibitor with a MEK inhibitor led to a synergistic anti-proliferation effect in MM cell lines. Thus, our study provides a rationale for future clinical trials of temsirolimus in MM patients as a component of combination therapy and suggests that MM cases with a high p-S6 but low p-AKT level could benefit from this combination. This was irrespective of cytogenetic features and reveals an additional way to classify MM cells. Multiple myeloma (MM) is an incurable, malignant B cell disorder characterized by frequent relapses and a poor prognosis. Thus, new therapeutic approaches are warranted. The phosphatidylinositol-3-kinase (PI3K) pathway plays a key role in many critical cellular processes, including cell proliferation and survival. Activated PI3K/AKT (protein kinases B)/mTOR (mammalian target of rapamycin) signaling has been identified in MM primary patient samples and cell lines. In this study, the efficacy of PI3K and mTOR inhibitors in various MM cell lines representing three different prognostic subtypes was tested. Whereas MM cell lines were rather resistant to PI3K inhibition, treatment with the mTOR inhibitor temsirolimus decreases the phosphorylation of key molecules in the PI3K pathway in MM cell lines, leading to G0/G1 cell cycle arrest and thus reduced proliferation. Strikingly, the efficacy of temsirolimus was amplified by combining the treatment with the Mitogen-activated protein kinase kinase (MEK) inhibitor trametinib. Our findings provide a scientific rationale for the simultaneous inhibition of mTOR and MEK as a novel strategy for the treatment of MM. [ABSTRACT FROM AUTHOR]

ThezincfingerproteinGrowthFactor Independence1 (GFI1) actsasatranscriptional repressor regulating differentiation of myeloid and lymphoid cells. A single nucleotide polymorphism of GFI1, GFI1-36N, has a prevalence of 7% in healthy Caucasiansand15%in acutemyeloidleukemia (AML)patients, hencemostprobably predisposing to AML. One reason for this is that GFI1-36N differs from thewildtype form GFI1-36S regarding its ability to induce epigenetic changes resulting in a derepression of oncogenes. Using proteomics, immunofluorescence, and immunoblotting we have now gained evidence that murine GFI1-36N leukemic cells exhibit a higher protein level of the pro-proliferative protein arginine Nmethyltransferase 5 (PRMT5) as well as increased levels of the cell cycle propagating cyclin-dependent kinases 4 (CDK4) and 6 (CDK6) leading to a faster proliferation of GFI1-36N leukemic cells in vitro. As a therapeutic approach, we subsequently treated leukemic GFI1-36S and GFI1-36N cells with the CDK4/6 inhibitor palbociclib and observed that GFI1-36N leukemic cells were more susceptible to this treatment. The findings suggest that presence of the GFI1-36N variant increases proliferation of leukemic cells and could possibly be amarker for a specific subset of AML patients sensitive to CDK4/6 inhibitors such as palbociclib. [ABSTRACT FROM AUTHOR]

Summary: Acute myeloid leukaemia (AML) is a haematological malignancy characterized by a poor prognosis. Bone marrow mesenchymal stromal cells (BM MSCs) support leukaemic cells in preventing chemotherapy‐induced apoptosis. This encouraged us to investigate leukaemia‐BM niche‐associated signalling and to identify signalling cascades supporting the interaction of leukaemic cells and BM MSC. Our study demonstrated functional differences between MSCs originating from leukaemic (AML MSCs) and healthy donors (HD MSCs). The direct interaction of leukaemic and AML MSCs was indispensable in influencing AML cell proliferation. We further identified an important role for Notch expression and its activation in AML MSCs contributing to the enhanced proliferation of AML cells. Supporting this observation, overexpression of the intracellular Notch domain (Notch ICN) in AML MSCs enhanced AML cells' proliferation. From a therapeutic point of view, dexamethasone treatment impeded Notch signalling in AML MSCs resulting in reduced AML cell proliferation. Concurrent with our data, Notch inhibitors had only a marginal effect on leukaemic cells alone but strongly influenced Notch signalling in AML MSCs and abrogated their cytoprotective function on AML cells. In vivo, dexamethasone treatment impeded Notch signalling in AML MSCs leading to a reduced number of AML MSCs and improved survival of leukaemic mice. In summary, targeting the interaction of leukaemic cells and AML MSCs using dexamethasone or Notch inhibitors might further improve treatment outcomes in AML patients. [ABSTRACT FROM AUTHOR]

Cytomegalovirus (HCMV) reactivation occurs frequently after hematopoietic stem cell transplantation and is associated with an increased treatment-related mortality. Induction of apoptosis by HCMV is unusual because HCMV utilizes various strategies to prevent apoptosis in infected cells in order to delay cell death and maintain viral replication. Here we show that HCMV can infect the acute leukemia cell lines Kasumi-1 (AML) and SD-1 (BCR-ABL-positive ALL), which inhibited their proliferation and induced apoptosis in almost all cells after 14 days. Although HCMV induced a significant up-regulation of the anti-apoptotic genecFLIPand the anti-stress geneGadd45a,and simultaneously down-regulated the pro-apoptotic genesp53, Gadd45gammain Kasumi-1 and SD-1 cells, we found that these anti-apoptotic mechanisms failed in HCMV-infected acute leukemia cells and apoptosis occurred via a caspase-dependent pathway. We conclude that HCMV can provide anti-leukemic effectsin vitro. To determine if this phenomenon may be clinically relevant further investigations will be required. [ABSTRACT FROM AUTHOR]

Objective: Retroviral vectors represent one of the most robust technologies for in vivo expression of heterologous gene sequences and are still the most commonly used vectors in clinical gene therapy trials. The production of high titer retroviral preparations, however, can be a problematic procedure for certain constructs. Methods: GALV- or RD114-pseudotyped retroviral particles carrying selectable fluorescence markers or drug resistance genes, such as the green fluorescent protein (GFP) or the O6-methylguanine-DNA-methyltransferase (MGMT) mutants, were used to stably transduce Phoenix-(FNX-)eco cells. Thereafter, a polyclonal population of producer cells was generated by enriching cells with high marker gene expression. In addition, single producer clones were selected by limiting dilution. Results: Retroviral titers were increased 1–2 logs by enriching for a polyclonal population of producer cells, and selection of single producer clones allowed another 1- to 2-log increase in titers. Using this method, reproducibly high titer viral preparations allowing efficient transduction of hematopoietic stem cells were generated. Conclusion: A reliable and time-effective method to generate stable high titer producer cells based on the FNX-cell line for problematic retroviral vector constructs is described. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The present study evaluated cellular and humoral immune parameters in myeloma patients, focusing on the effect of treatment and the risk of opportunistic infections. Peripheral blood lymphocyte subsets and serum levels of nonmyeloma immunoglobulins (Ig) were analysed in 480 blood samples from 77 myeloma patients. Untreated myeloma patients exhibited significantly reduced CD4+/45RO+, CD19+, CD3+/HLA-DR+, and natural killer (NK) cells, as well as nonmyeloma IgA, IgG and IgM. Conventional-dose chemotherapy resulted in significantly reduced CD4+ and even further decline of CD4+/CD45RO+ and CD19+ cells, most notably in relapsed patients. Additional thalidomide treatment had no significant effects on these parameters. Following high-dose chemotherapy (HD-CTX), prolonged immunosuppression was observed. Although CD8+, NK, CD19+ and CD+/CD45RO+ cells recovered to normal values within 60, 90, 360 and 720 days, respectively, CD4+ counts remained reduced even thereafter. Nine opportunistic infections were observed, including five cytomegalovirus (CMV) diseases, one Pneumocystis carinii pneumonia (PCP) and three varicella zoster virus infections with CMV diseases and PCP occurring exclusively after HD-CTX. Opportunistic infections were correlated with severely reduced CD4+, as well as CD4+/CD45RO+ and CD19+ counts. Thus, myeloma patients display cellular and humoral immunodeficiencies, which increase following conventional as well as HD-CTX, and constitute an important predisposing factor for opportunistic infections. [ABSTRACT FROM AUTHOR]