Preferential transcription of the mutated allele in NPM1 mutated acute myeloid leukaemia.

Nucleophosmin is commonly both over-expressed and mutated in acute myeloid leukemia (AML). NPM1 mutations are always heterozygous. In addition, NPM1 has a number of different splice variants with the major variant encoded by exons 1-9 and 11-12 (NPM1.1). Further variants include NPM1.2 which lacks exons 8 and 10 and NPM1.3 which comprises exons 1-10 (and so lacks the region of sequence mutated in AML). In this study we quantified the expression of these three variants in 108 AML patient samples with and without NPM1 mutations and also assessed the level of expression from the wild-type and mutant alleles in variants NPM1.1 and NPM1.2. The results show that NPM1.1 is the most commonly expressed variant, however transcripts from wild-type and mutated alleles do not occur at equal levels, with a significant bias toward the mutated allele. Considering the involvement of mutant nucleophosmin in the progression and maintenance of AML, a bias towards mutated transcripts could have a significant impact on disease maintenance.

Caspase-8 polymorphisms result in reduced Alemtuzumab-induced T-cell apoptosis and worse survival after transplantation.

Allo-SCT using unrelated donors is a curative treatment for patients with hematological disorders. The best donor is one matched for 10/10 HLA alleles, however studies have shown an additional survival benefit when considering other genetic factors. It has been shown that a six-nucleotide insertion/deletion polymorphism in the CASP8 gene promoter results in reduced susceptibility of T lymphocytes to undergo apoptosis. In 186 SCT recipients, we found a significantly better OS in those who received a transplant from a WT/WT donor compared with donors with a deletion (3 years: 52 vs 34%; P=0.03; multivariate analysis; RR 0.61; 95% CI 0.38-0.98, P=0.04). This was more marked when both the patient and the donor had a deletion (3 years OS: 62% compared with 36%, P=0.01). As the majority of these patients received Alemtuzumab during conditioning, we went on to analyze the in vitro effect of the polymorphism on Alemtuzumab-induced apoptosis. We showed statistically significantly higher percentages of apoptotic naïve CD4 (P<0.0005) and CD8 (P<0.0005) T cells in WT/WT donors in comparison with donors with a deletion. These data imply an unrecognized role for the CASP8 promoter polymorphism on survival following unrelated SCT particularly in the context of T-cell depletion with Alemtuzumab.

Analysis of factors that affect in vitro chemosensitivity of leukaemic stem and progenitor cells to gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukaemia.

Relapse in acute myeloid leukaemia (AML) is considered to result from the persistence of drug-resistant leukaemic stem and progenitor cells (LSPC) within a bone marrow 'niche' microenvironment. Identifying novel agents that have the potential to target these LSPC in their niche microenvironment will aid in the characterization of candidate agents for post-remission chemotherapy. Using an in vitro model, we found that 48-h culture with gemtuzumab ozogamicin (Mylotarg) resulted in a 34% reduction in CD34(+)CD38(-)CD123(+) LSPC number, whereas normal CD34(+)CD38(-) haemapoietic stem cells were insensitive to this agent. As there was considerable heterogeneity in LSPC response to Mylotarg treatment, various factors potentially underpinning the differential response were assessed. LSPC that overexpressed CD33 (P=0.01), which were P-glycoprotein-negative (P=0.008) and with internal tandem duplication (ITD) of the FLT3 gene (FLT3/ITD) status (P=0.006) responded better to Mylotarg treatment. LSPC from patient samples that have these combined characteristics as well as low LSPC burden showed significantly more chemosensitivity to Mylotarg compared with all other cases (P=0.002). In multivariate analysis, LSPC burden and FLT3 status were found to be predictors of LSPC chemosensitivity to Mylotarg treatment (P<0.0001). In conclusion, we have shown heterogeneity in the LSPC compartment of AML patients underpinning differential in vitro sensitivity to Mylotarg.

Resistance to FLT3 inhibition in an in vitro model of primary AML cells with a stem cell phenotype in a defined microenvironment.

Relapse in acute myeloid leukaemia (AML) is mediated by survival of leukaemic stem cells following remission-induction chemotherapy. It would therefore be useful to identify therapeutic agents that target leukaemic stem cells. We devised a flow cytometric chemosensitivity assay allowing 48 h culture of leukaemic blasts in a defined microenvironment followed by enumeration of viable CD34+CD38-CD123+ leukaemic stem and progenitor cells (LSPC). The assay was used to investigate the LSPC response to cytosine arabinoside (Ara-C) and to the FLT3 inhibitor AG1296. There was a 3.6-fold increase in Ara-C-treated LSPC survival under defined 'niche-like' conditions compared to culture without microenvironmental support. Nine AML samples with internal tandem duplications of FLT3 (FLT3/ITDs) were treated with AG1296. Three samples were very sensitive (>50% kill) and 4 were moderately sensitive (10-50% kill) in bulk suspension culture without microenvironmental support. However, under defined 'niche-like' conditions, the survival of LSPC was enhanced rather than inhibited by AG1296 treatment. We conclude that an interaction between LSPC and a defined in vitro microenvironment models a chemoresistant niche. Our data point to a need to investigate more novel chemotherapeutic agents under these stringent conditions to identify agents that may be suitable to target minimal residual disease in AML.

DNA repair contributes to the drug-resistant phenotype of primary acute myeloid leukaemia cells with FLT3 internal tandem duplications and is reversed by the FLT3 inhibitor PKC412.

The presence of internal tandem duplications (ITD) mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor influences the risk of relapse in acute myeloid leukaemia (AML). We have investigated DNA repair in FLT3-ITD and wild-type (WT) cells. Using the comet assay, we have demonstrated that the FLT3 inhibitor PKC412 significantly inhibits repair of DNA damage in the MV4-11-FLT3-ITD cell line and FLT3-ITD patient samples but not in the HL-60-FLT3-WT cell line or FLT3-WT patient samples. Following the discovery that transcript levels of the DNA repair gene RAD51 are significantly correlated with FLT3 transcript levels in FLT3-ITD patients, we further investigated the role of RAD51 in FLT3-ITD-AML. The reduction in DNA repair in PKC412-treated FLT3-ITD cells was shown to be associated with downregulation of RAD51 mRNA and protein expression and correlates with the maintenance of phosphorylated H2AX levels, implying that PKC412 inhibits the homologous recombination double-strand break repair pathway in FLT3-ITD cells. Using FLT3-short interfering RNA (siRNA), we also demonstrated that genetic silencing of FLT3 results in RAD51 downregulation in FLT3-ITD cells but not in FLT3-WT cells. This work suggests that the use of FLT3 inhibitors such as PKC412 may reverse the drug-resistant phenotype of FLT3-ITD-AML cells by inhibiting repair of chemotherapy-induced genotoxic damage and thereby reduce the risk of disease relapse.

Methylation of the hMLH1 promoter and its association with microsatellite instability in acute myeloid leukemia.

The hMLH1 and hMSH2 genes are involved in the DNA mismatch repair (MMR) pathway. Defects in either of these genes have been associated with genetic instability in a wide variety of malignancies. A molecular mechanism involved in aberrant MMR gene expression is the epigenetic silencing of transcription by promoter methylation. The importance of MMR promoter methylation in leukemia is presently unclear and we have therefore undertaken a detailed analysis of the promoter regions of hMLH1 and hMSH2 using the technique of bisulfite genomic sequencing. DNA from 55 patients with acute myeloid leukemia (AML) including 23 patients with therapy-related AML (t-AML) have been analyzed. Two patients with de novo AML demonstrated extensive methylation throughout the whole hMLH1 region sequenced, one of whom had previously shown widespread genetic instability, measured as microsatellite instability (MSI). However methylation of hMLH1 was not found in t-AML which has previously been associated with MSI. In addition, methylation was seen at a restricted region of the hMLH1 promoter in both AML patients and healthy controls. The significance of this methylated region of the hMLH1 promoter is uncertain, however, our results confirm that in some patients with AML extensive methylation of hMLH1, but not of hMSH2 may occur, and as is the case in solid tumors this can be associated with the presence of a defective DNA mismatch repair pathway resulting in MSI.

Microsatellite instability occurs in defined subsets of patients with acute myeloblastic leukaemia.

Using a sensitive fluorescent-polymerase chain reaction technique we looked for microsatellite instability (MSI) as functional evidence of mismatch repair defects in 71 cases of acute myeloblastic leukaemia (AML). MSI was assessed at 11 loci in matched leukaemic and constitutional DNA. Nine out of 71 patients (13%) were found to have MSI. Four of these patients had therapy-related leukaemia and the remaining five were all over the age of 60 years. There was a high incidence of adverse-risk cytogenetics in the patients with MSI, including abnormalities of chromosomes 5 and/or 7. Of the nine cases of t-AML included in this study, four (44%) had MSI. MSI was also seen in five of 51 cases (10%) over the age of 60 years but not in any cases under the age of 60 years with de novo AML. Using a sensitive assay, our results suggest that MSI occurs in two subgroups of patients with AML: those with t-AML and the elderly (> 60 years), but is rare in younger patients.

Anti-8-oxo-2'-deoxyguanosine phage antibodies: isolation, characterization, and relationship to disease states.

We have used human single chain Fv (scFv) phage display antibody libraries to isolate recombinant antibodies against the DNA adduct 8-oxo-2'-deoxyguanosine (8-oxodG). One of these scFvs (175G) bound to several 8-oxodG-containing oligonucleotides whilst demonstrating no cross-reactivity with G-containing control oligonucleotides, and bound to 8-oxodG lesions introduced into DNA by treatment with methylene blue and white light. In addition, 175G inhibited the cleavage of an 8-oxodG-containing oligonucleotide by the Escherichia coli enzyme formamidopyrimidine-DNA glycosylase (Fpg). The nucleotide sequence of the 175G V(H) gene segment was 98% homologous to the published V(H) sequence of a human hybridoma derived from a patient with systemic lupus erythematosus (SLE). Sera from two SLE patients bound to damaged DNA, and this binding could be inhibited by 175G. The use of human scFv phage display libraries has thus produced a unique reagent with specificity for 8-oxodG, which may have a role in damage detection and quantitation and in modifying DNA repair activity. 175G also offers support to the hypothesis that SLE might be associated with oxidative damage to DNA.

DNA repair mechanisms and acute myeloblastic leukemia.

DNA repair mechanisms play a vital role in maintaining genomic integrity. With the wealth of knowledge gained recently on these processes it is becoming clear that defects in repair proteins and proteins associated with the regulation of repair are connected to many different human diseases including cancer. This paper has aimed to review the four major DNA repair processes and in particular concentrate on their association with acute myeloblastic leukemia (AML).