Deregulation of Mitochondrial ATPsyn-β in Acute Myeloid Leukemia Cells and with Increased Drug Resistance

The mechanisms underlying the development of multidrug resistance in acute myeloid leukemia are not fully understood. Here we analyzed the expressions of mitochondrial ATPsyn-β in adriamycin-resistant cell line HL-60/ADM and its parental cell line HL-60. Meanwhile we compared the differences of mitochondrial ATPsyn-β expression and ATP synthase activity in 110 acute myeloid leukemia (AML, non-M3) patients between relapsed/refractory and those in remission. Our results showed that down-regulation of ATPsyn-β expression by siRNA in HL-60 cells increased cell viability and apoptotic resistance to adriamycin, while up-regulation of mitochondrial ATPsyn-β in HL-60/ADM cells enhanced cell sensitivity to adriamycin and promoted apoptosis. Mitochondrial ATPsyn-β expression and ATP synthase activity in relapsed/refractory acute myeloid leukemia patients were downregulated. This downregulated ATPsyn-β expression exhibited a positive correlation with the response to adriamycin of primary cells. A lower expression of ATPsyn-β in newly diagnosed or relapsed/refractory patients was associated with a shorter first remission duration or overall survival. Our findings show mitochondrial ATPsyn-β plays an important role in the mechanism of multidrug resistance in AML thus may present both a new marker for prognosis assessment and a new target for reversing drug resistance.     

Engagement of SIRPα Inhibits Growth and Induces Programmed Cell Death in Acute Myeloid Leukemia Cells

Background

Recent studies show the importance of interactions between CD47 expressed on acute myeloid leukemia (AML) cells and the inhibitory immunoreceptor, signal regulatory protein-alpha (SIRPα) on macrophages. Although AML cells express SIRPα, its function has not been investigated in these cells. In this study we aimed to determine the role of the SIRPα in acute myeloid leukemia.

Design and Methods

We analyzed the expression of SIRPα, both on mRNA and protein level in AML patients and we further investigated whether the expression of SIRPα on two low SIRPα expressing AML cell lines could be upregulated upon differentiation of the cells. We determined the effect of chimeric SIRPα expression on tumor cell growth and programmed cell death by its triggering with an agonistic antibody in these cells. Moreover, we examined the efficacy of agonistic antibody in combination with established antileukemic drugs.

Results

By microarray analysis of an extensive cohort of primary AML samples, we demonstrated that SIRPα is differentially expressed in AML subgroups and its expression level is dependent on differentiation stage, with high levels in FAB M4/M5 AML and low levels in FAB M0–M3. Interestingly, AML patients with high SIRPα expression had a poor prognosis. Our results also showed that SIRPα is upregulated upon differentiation of NB4 and Kasumi cells. In addition, triggering of SIRPα with an agonistic antibody in the cells stably expressing chimeric SIRPα, led to inhibition of growth and induction of programmed cell death. Finally, the SIRPα-derived signaling synergized with the activity of established antileukemic drugs.

Conclusions

Our data indicate that triggering of SIRPα has antileukemic effect and may function as a potential therapeutic target in AML.     

Stabilization of NF-κB-Inducing Kinase Suppresses MLL-AF9-Induced Acute Myeloid Leukemia

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Gene therapy in the clinic: whose risks?

Current experience with gene therapy for X-linked severe combined immunodeficiency disorder (X-SCID) suggests that we might now be at the point where it can be considered the 'standard of care'. This therapy carries an unquantified risk of leukaemia, raising important questions for regulators. How dangerous does a potentially curative therapy for a fatal illness need to be before we call it unsafe. How uncertain does a risk need to be for us to regard a therapy as still 'experimental'? Whose interests should prevail? Here I argue that in X-SCID it is the parents and children whom we should listen to first and foremost. How far does this patient-centred approach to licensing therapies extend? Can it be given a rational basis?     

BCR-ABL Kinase Domain Mutations in Chronic Myeloid Leukemia: Not Quite Enough to Cause Resistance to Imatinib Therapy?

Patients with chronic myeloid leukemia (CML) treated with imatinib in early chronic phase tend to have durable remissions, but there is a high rate of relapse in patients with advanced disease. Mutations in the kinase domain of BCR-ABL that impair drug binding have been identified as the major mechanism of resistance. It is not known when exactly these mutations arise, but in some patients retrospective analysis of pretherapeutic samples demonstrated identical mutations, suggesting selection in the presence of drug. In the present study we have used a highly sensitive PCR assay to screen for kinase domain mutations in pretherapeutic samples from CML patients, irrespective of their subsequent response to imatinib. We find that kinase domain mutations are demonstrable in approximately 1/3 of patients with accelerated phase or blast crisis and that the presence of 2 copies of the Philadelphia chromosome is strongly correlated with mutation detection. Unexpectedly, kinase domain mutant clones were not invariably selected in the presence of drug, suggesting that additional mechanisms must contribute to a fully drug resistant leukemia.     

Feasibility of Azacitidine Added to Standard Chemotherapy in Older Patients with Acute Myeloid Leukemia — A Randomised SAL Pilot Study

Introduction

Older patients with acute myeloid leukemia (AML) experience short survival despite intensive chemotherapy. Azacitidine has promising activity in patients with low proliferating AML. The aim of this dose-finding part of this trial was to evaluate feasibility and safety of azacitidine combined with a cytarabine- and daunorubicin-based chemotherapy in older patients with AML.

Trial Design

Prospective, randomised, open, phase II trial with parallel group design and fixed sample size.

Patients and Methods

Patients aged 61 years or older, with untreated acute myeloid leukemia with a leukocyte count of <20,000/µl at the time of study entry and adequate organ function were eligible. Patients were randomised to receive azacitidine either 37.5 (dose level 1) or 75 mg/sqm (dose level 2) for five days before each cycle of induction (7+3 cytarabine plus daunorubicine) and consolidation (intermediate-dose cytarabine) therapy. Dose-limiting toxicity was the primary endpoint.

Results

Six patients each were randomised into each dose level and evaluable for analysis. No dose-limiting toxicity occurred in either dose level. Nine serious adverse events occurred in five patients (three in the 37.5 mg, two in the 75 mg arm) with two fatal outcomes. Two patients at the 37.5 mg/sqm dose level and four patients at the 75 mg/sqm level achieved a complete remission after induction therapy. Median overall survival was 266 days and median event-free survival 215 days after a median follow up of 616 days.

Conclusions

The combination of azacitidine 75 mg/sqm with standard induction therapy is feasible in older patients with AML and was selected as an investigational arm in the randomised controlled part of this phase-II study, which is currently halted due to an increased cardiac toxicity observed in the experimental arm.

Trial Registration

This trial is registered at clinical trials.gov (identifier: {"type":"clinical-trial","attrs":{"text":"NCT00915252","term_id":"NCT00915252"}}NCT00915252).     

Gene Regulation in N Mutants of Bacteriophage λ

Mutants (N(-)nin) of bacteriophage lambda in which the N gene product is not required for growth on wild-type Escherichia coli do not plate on recA bacterial mutants. Secondary mutants, selected for growth on recA, lie within the immunity region to the right of gene cI and appear identical to the cro mutants of Eisen et al. In an N(+) phage, a cro mutation causes enhanced and prolonged production of lambda exonuclease. N(-)cro phages make no detectable exonuclease, but show an increased rate of specific excision from lysogens and are excluded by P2 prophage. These properties, together with the ability to plate on recA, suggest that N(-)cro phages express genes to the left of N at a rate that is very low but higher than that for N(-)cro(+) phages. N(-)nin phages can integrate at the normal site on the bacterial chromosome, but specific excision from lysogens is immeasurably low.     

Prognostic Relevance of Cytokine Receptor Expression in Acute Myeloid Leukemia: Interleukin-2 Receptor α-Chain (CD25) Expression Predicts a Poor Prognosis

A variety of cytokine/cytokine receptor systems affect the biological behavior of acute leukemia cells. However, little is known about the clinical relevance of cytokine receptor expression in acute myeloid leukemia (AML). We quantitatively examined the expression of interleukin-2 receptor α-chain (IL-2Rα, also known as CD25), IL-2Rβ, IL-3Rα, IL-4Rα, IL-5Rα, IL-6Rα, IL-7Rα, the common β-chain (βc), γc, granulocyte-macrophage colony-stimulating factor (GM-CSF)Rα, G-CSFR, c-fms, c-mpl, c-kit, FLT3, and GP130 in leukemia cells from 767 adult patients with AML by flow cytometry and determined their prevalence and clinical significance. All cytokine receptors examined were expressed at varying levels, whereas the levels of IL-3Rα, GM-CSFRα, IL-2Rα, γc, c-kit, and G-CSFR exhibited a wide spectrum of ≥10,000 sites/cell. In terms of their French-American-British classification types, GM-CSFRα and c-fms were preferentially expressed in M4/M5 patients, G-CSF in M3 patients, and IL-2Rα in non-M3 patients. Elevated levels of IL-3Rα, GM-CSFRα, and IL-2Rα correlated with leukocytosis. In patients ≤60 years old, higher levels of these 3 receptors correlated with poor responses to conventional chemotherapy, but only IL-2Rα was associated with a shorter overall survival. By incorporating IL-2Rα status into cytogenetic risk stratification, we could sort out a significantly adverse-risk cohort from the cytogenetically intermediate-risk group. Analyses with various phenotypical risk markers revealed the expression of IL-2Rα as an independent prognostic indicator in patients with intermediate-risk cytogenetics. These findings were not observed in patients >60 years old. Our results indicate that several cytokine receptors were associated with certain cellular and clinical features, but IL-2Rα alone had prognostic value that provides an additional marker to improve current risk evaluation in AML patients ≤60 years old.     

Stochastic Modeling of Gene Regulation by Noncoding Small RNAs in the Strong Interaction Limit

Noncoding small RNAs (sRNAs) are known to play a key role in regulating diverse cellular processes, and their dysregulation is linked to various diseases such as cancer. Such diseases are also marked by phenotypic heterogeneity, which is often driven by the intrinsic stochasticity of gene expression. Correspondingly, there is significant interest in developing quantitative models focusing on the interplay between stochastic gene expression and regulation by sRNAs. We consider the canonical model of regulation of stochastic gene expression by sRNAs, wherein interaction between constitutively expressed sRNAs and mRNAs leads to stoichiometric mutual degradation. The exact solution of this model is analytically intractable given the nonlinear interaction term between sRNAs and mRNAs, and theoretical approaches typically invoke the mean-field approximation. However, mean-field results are inaccurate in the limit of strong interactions and low abundances; thus, alternative theoretical approaches are needed. In this work, we obtain analytical results for the canonical model of regulation of stochastic gene expression by sRNAs in the strong interaction limit. We derive analytical results for the steady-state generating function of the joint distribution of mRNAs and sRNAs in the limit of strong interactions and use the results derived to obtain analytical expressions characterizing the corresponding protein steady-state distribution. The results obtained can serve as building blocks for the analysis of genetic circuits involving sRNAs and provide new insights into the role of sRNAs in regulating stochastic gene expression in the limit of strong interactions.     

Gene transfer in bacteria: speciation without species?

Although Bacteria and Archaea reproduce by binary fission, exchange of genes among lineages has shaped the diversity of their populations and the diversification of their lineages. Gene exchange can occur by two distinct routes, each differentially impacting the recipient genome. First, homologous recombination mediates the exchange of DNA between closely related individuals (those whose sequences are sufficient similarly to allow efficient integration). As a result, homologous recombination mediates the dispersal of advantageous alleles that may rise to high frequency among genetically related individuals via periodic selection events. Second, lateral gene transfer can introduce novel DNA into a genome from completely unrelated lineages via illegitimate recombination. Gene exchange by this route serves to distribute genes throughout distantly related clades and therefore may confer complex abilities--not otherwise found among closely related lineages--onto the recipient organisms. These two mechanisms of gene exchange play complementary roles in the diversification of microbial populations into independent, ecologically distinct lineages. Although the delineation of microbial "species" then becomes difficult--if not impossible--to achieve, a cogent process of speciation can be predicted.