Targeting of CD34+CD38- cells using Gemtuzumab ozogamicin (Mylotarg) in combination with tipifarnib (Zarnestra) in Acute Myeloid Leukaemia.

BACKGROUND: The CD34+CD38- subset of AML cells is enriched for resistance to current chemotherapeutic agents and considered to contribute to disease progression and relapse in Acute Myeloid Leukaemia (AML) patients following initial treatment. METHODS: Chemosensitivity in phenotypically defined subsets from 34 primary AML samples was measured by flow cytometry following 48 hr in vitro treatment with gemtuzumab ozogamicin (GO, Mylotarg) and the farnesyltransferase inhibitor tipifarnib/zarnestra. The DNA damage response was measured using flow cytometry, immunofluorescence and immunohistochemistry. RESULTS: Using a previously validated in vitro minimal residual disease model, we now show that the combination of GO (10 ng/ml) and tipifarnib (5 µM) targets the CD34+CD38- subset resulting in 65% median cell loss compared to 28% and 13% CD34+CD38- cell loss in GO-treated and tipifarnib-treated cells, respectively. Using phosphokinome profiling and immunofluorescence in the TF-1a cell line, we demonstrate that the drug combination is characterised by the activation of a DNA damage response (induction of γH2A.X and thr68 phosphorylation of chk2). Higher induction of γH2AX was found in CD34+CD38- than in CD34+CD38+ patient cells. In a model system, we show that dormancy impairs damage resolution, allowing accumulation of γH2AX foci. CONCLUSIONS: The chemosensitivity of the CD34+CD38- subset, combined with enhanced damage indicators, suggest that this subset is primed to favour programmed cell death as opposed to repairing damage. This interaction between tipifarnib and GO suggests a potential role in the treatment of AML.

QTL analyses of lineage-negative mouse bone marrow cells labeled with Sca-1 and c-Kit.

Differences in the number of functionally and/or phenotypically defined bone marrow cells in inbred mouse strains have been exploited to map quantitative trait loci (QTL) that determine the variation in cell frequency. To extend this approach to the differences in the stem/progenitor cell compartment in CBA/H and C57BL/6 mice, we have exploited the resolution of flow cytometry and the power of QTL analyses in 124 F(2) mice to analyze lineage-negative (Lin(-)) bone marrow cells according to the intensity of labeling with Sca-1 and c-Kit. In the Lin(-) Sca-1(+) c-Kit(+) enriched population, six QTL were identified: one significant and five suggestive. Whereas previous in vitro clonogenic, LTC-IC, day 35 CAFC, and flow cytometry each identified different QTL, our approach identified the same or very similar QTL at all three loci (chromosomes 1, 17, and 18) as well as QTL on chromosomes 6 and 10. In silico analyses implicate hematopoietic stem cell homing involving Cxcr4 and Cxcl12 as being the determining pathway. The mapping of the same or very similar QTL in independent studies using different assay(s) suggests a common genetic determinant, and thus reinforces the biological and genetic significance of the QTL. These data also suggest that mouse bone marrow cell subpopulations can be functionally, phenotypically, and genetically defined.

DNA methylation during mouse hemopoietic differentiation and radiation-induced leukemia.

OBJECTIVE: To examine DNA methylation in mouse hemopoiesis before and after in vivo exposure to a leukemogenic dose of x-rays, and address whether methylation levels are associated with the relative radiosensitivity of tissues in vivo. METHODS: The methylation status of control CBA/H and C57BL/6 mouse tissues before and after exposure to 3-Gy x-rays, and myeloid and lymphoid leukemias and lymphomas, was assessed by the direct analysis of the 5-methylcytosine (5-(Me)C) content of DNA, and by Southern blot analysis of genomic repeat sequences. RESULTS: The DNA 5-(Me)C content of bone marrow is 15% lower than spleen. Together with the analyses of stem (myeloid) and progenitor (lymphoid) leukemias and lymphomas, we found a trend of increasing methylation during hemopoietic differentiation. Exposure to x-rays induced greater cell death in the hypomethylated bone marrow (>80%) than spleen (50%) in vivo, supporting the observed correlation found between methylation status and radiosensitivity of other high-turnover hierarchical tissues. Furthermore, there was an 8% DNA 5-(Me)C content decrease in bone marrow after in vivo exposure to 3-Gy x-rays, but this was genotype dependent, being observed in AML-susceptible (CBA/H) but not AML-resistant (C57BL/6) inbred mice. CONCLUSION: Together these data suggest that methylation status may be related to the relative radiosensitivity of high-turnover hierarchical tissues such as bone marrow and that radiation-induced DNA hypomethylation has a role in radiation leukemogenesis.

Polymorphisms in human homeobox HLX1 and DNA repair RAD51 genes increase the risk of therapy-related acute myeloid leukemia.

Studies of radiation-induced acute myeloid leukemia (AML) in mice suggest that the number of target stem cells is a risk factor, and the HLX1 homeobox gene, which is important for hematopoietic development, is a candidate gene. The distribution of the C/T-3' untranslated region (UTR) polymorphism in HLX1 in patients with AML and therapy-related AML (t-AML) compared with controls was therefore determined. The presence of the variant HLX1 allele significantly increases the risk of t-AML (OR = 3.36, 95% CI, 1.65-6.84). The DNA repair gene RAD51 (135G/C-5' UTR) polymorphism also increases t-AML risk, and when combined analysis was performed on both RAD51 and HLX1 variant alleles, a synergistic 9.5-fold increase (95% CI, 2.22-40.64) in the risk of t-AML was observed. We suggest that the HLX1 polymorphism has an effect on stem cell numbers, whereas an increased DNA repair capacity (RAD51) will suppress apoptosis, a genetic interaction that may increase the number of genomes at risk during cancer therapy.

Evidence for clustered tumour suppressor gene loci on mouse chromosomes 2 and 4 in radiation-induced acute myeloid leukaemia.

PURPOSE: To investigate the influence of genetic and epigenetic factors on allelic loss on chromosomes 2 and 4 in mouse radiation-induced acute myeloid leukaemia (r-AML). METHODS: r-AML that arose in (CBA/HxC57BL/6)F1xCBA/H and F1xC57BL/6 mice were screened for transcription factor PU1 (also known as SPI-1) gene mutations and methylation of the paired box gene 5 (Pax5) gene promoter. We have increased the statistical significance of a genetic linkage analysis of affected F1xCBA/H mice to test for linkage to loci implicated directly or indirectly with r-AML-susceptibility. RESULTS: There was a statistically significant difference ( p < 10-4) in the frequency of PU1 gene mutations in F1xCBA/H and F1xC57BL/6 r-AML, implicating a second linked but genotype-dependent myeloid leukaemia suppressor gene on chromosome 2. A suggestive CBA/H r-AML-resistance locus maps within 10 cM of the minimally deleted region on chromosome 4. The Pax5 gene promoter is subject to ongoing subclonal promoter methylation in the r-AML, evidence that Pax5 gene silencing confers a selective advantage during clonal expansion in vivo. CONCLUSIONS: Allelic loss in mouse r-AML and subsequent tumour suppressor gene mutation (PU1) or silencing (Pax5) is strongly influenced by genetic background and/or epigenetic factors, and driven by in vivo clonal selection.

A cryptic t(5;11)(q35;p15.5) in 2 children with acute myeloid leukemia with apparently normal karyotypes, identified by a multiplex fluorescence in situ hybridization telomere assay.

The identification of specific chromosome abnormalities in acute myeloid leukemia (AML) is important for the stratification of patients into the appropriate treatment protocols. However, a significant proportion of diagnostic bone marrow karyotypes in AML is reported as normal by conventional cytogenetic analysis and it is suspected that these karyotypes may conceal the presence of diagnostically significant chromosome rearrangements. To address this question, we have developed a novel 12-color fluorescence in situ hybridization (FISH) assay for telomeric rearrangements (termed M-TEL), which uses an optimized set of chromosome-specific subtelomeric probes. We report here the application of the M-TEL assay to 69 AML cases with apparently normal karyotypes or an isolated trisomy. Of the 69 cases examined, 3 abnormalities were identified, all in the normal karyotype group. The first was a t(11;19)(q23;p13), identified in an infant with AML-M4. In 2 other young patients with AML (< 19 years), an apparently identical t(5;11)(q35;p15.5) was identified. Breakpoint mapping by FISH and reverse transcriptase polymerase chain reaction (RT-PCR) analysis confirmed that this was the same t(5;11) as previously identified in 3 children with AML, associated with del(5q) and resulting in the NUP98-NSD1 gene fusion. The t(5;11) was not detected by 24-color karyotyping using multiplex FISH (M-FISH), emphasizing the value of screening with subtelomeric probes for subtle translocations. This is the first report of the t(5;11)(q35;p15.5) in association with an apparently normal karyotype, and highlights this as a new, potentially clinically significant chromosome rearrangement in childhood AML.