A phase I/II trial of AT9283, a selective inhibitor of aurora kinase in children with relapsed or refractory acute leukemia: challenges to run early phase clinical trials for children with leukemia.

Aurora kinases regulate mitosis and are commonly overexpressed in leukemia. This phase I/IIa study of AT9283, a multikinase inhibitor, was designed to identify maximal tolerated doses, safety, pharmacokinetics, and pharmacodynamic activity in children with relapsed/refractory acute leukemia. The trial suffered from poor recruitment and terminated early, therefore failing to identify its primary endpoints. AT9283 caused tolerable toxicity, but failed to show clinical responses. Future trials should be based on robust preclinical data that provide an indication of which patients may benefit from the experimental agent, and recruitment should be improved through international collaborations and early combination with established treatment strategies.

The role of the RAS pathway in iAMP21-ALL.

Intrachromosomal amplification of chromosome 21 (iAMP21) identifies a high-risk subtype of acute lymphoblastic leukaemia (ALL), requiring intensive treatment to reduce their relapse risk. Improved understanding of the genomic landscape of iAMP21-ALL will ascertain whether these patients may benefit from targeted therapy. We performed whole-exome sequencing of eight iAMP21-ALL samples. The mutation rate was dramatically disparate between cases (average 24.9, range 5-51) and a large number of novel variants were identified, including frequent mutation of the RAS/MEK/ERK pathway. Targeted sequencing of a larger cohort revealed that 60% (25/42) of diagnostic iAMP21-ALL samples harboured 42 distinct RAS pathway mutations. High sequencing coverage demonstrated heterogeneity in the form of multiple RAS pathway mutations within the same sample and diverse variant allele frequencies (VAFs) (2-52%), similar to other subtypes of ALL. Constitutive RAS pathway activation was observed in iAMP21 samples that harboured mutations in the predominant clone (⩾35% VAF). Viable iAMP21 cells from primary xenografts showed reduced viability in response to the MEK1/2 inhibitor, selumetinib, in vitro. As clonal (⩾35% VAF) mutations were detected in 26% (11/42) of iAMP21-ALL, this evidence of response to RAS pathway inhibitors may offer the possibility to introduce targeted therapy to improve therapeutic efficacy in these high-risk patients.

Sweet problems: insect traits defining the limits to dietary sugar utilisation by the pea aphid, Acyrthosiphon pisum.

Plant phloem sap is an extreme diet for animals, partly because of its high and variable sugar content. The physiological and feeding traits of the pea aphid Acyrthosiphon pisum that define the upper and lower limits to the range of dietary sucrose concentrations utilised by this insect were determined principally using chemically defined diets containing 0.125-1.5 mol l(-1) sucrose. On the diets with 0.125 mol l(-1) and 1.5 mol l(-1) sucrose, the aphids died as larvae within 8 and 14 days of birth, respectively. On the other diets, 60-96% of aphids developed to adulthood, and the 0.5 mol l(-1) and 0.75 mol l(-1) diets supported the highest fecundity. The diet with 0.125 mol l(-1) sucrose was ingested at 36% of the rate of the 0.25 mol l(-1) sucrose diet, but >90% of ingested sucrose-carbon was assimilated on both diets. This suggests that the lower limit is dictated by the aphid feeding response, specifically, a requirement for a minimal concentration of sucrose for sustained feeding. The haemolymph osmotic pressure of aphids on diets with 0.125-1.5 mol l(-1) sucrose was up to 68% higher than on 0.125-1.0 mol l(-1) sucrose diets, but diet consumption and sucrose-carbon assimilation was not reduced on the very high sucrose diets relative to 1.0 mol l(-1) sucrose. This suggests that failure of the osmoregulatory capacity of the insects on high sucrose diets may define the upper limit to the range of dietary sucrose utilised by the aphids. The mean haemolymph osmotic pressure of aphids on plants with phloem sap containing 0.37-0.97 mol l(-1) sucrose was 1.61+/-0.063 MPa (mean +/- s.e.m.), not significantly different from that (1.47+/-0.059 MPa) on diets with 0.25-1.0 mol l(-1) sucrose. It is concluded that the osmoregulatory response of aphids to diets and plants are comparable, and, more generally, that the feeding and osmoregulatory capabilities of the aphids are compatible with the phloem sugar levels commonly encountered by aphids feeding on plants.

The significance of gut sucrase activity for osmoregulation in the pea aphid, Acyrthosiphon pisum.

The osmotic pressure of the body fluids of aphids is lower than in their diet of plant phloem sap. It is hypothesised that aphids reduce the osmotic pressure of ingested food by sucrase-mediated hydrolysis of dietary sucrose to glucose and fructose, and the polymerisation of glucose into oligosaccharides of low osmotic pressure per hexose unit. To test this hypothesis, the impact of the alpha-glucosidase inhibitor acarbose on the sugar relations and osmoregulation of aphids was explored. Acarbose inhibited sucrase activity in gut homogenates and the production of monosaccharides and oligosaccharides in the honeydew of live aphids. Acarbose caused an increase in the haemolymph osmotic pressure for aphids reared on a diet (containing 0.75 M sucrose) hyperosmotic to the haemolymph and not on the isoosmotic diet containing 0.2 M sucrose. It did not affect aphid feeding rate over 2 days, except at high concentrations on 0.75 M sucrose diet, and this may have been a secondary consequence of osmotic dysfunction. Acarbose-treated aphids died prematurely. With 5 microM dietary acarbose, mean survivorship on 0.2 M sucrose diet was 4.2 days, not significantly different from starved aphids, indicating that, although these aphids fed, they were deprived of utilisable carbon; and on 0.75 M sucrose diet, mean survivorship was just 2.8 days, probably as a consequence of osmotic failure. It is concluded that the aphid gut sucrase activity is essential for osmoregulation of aphids ingesting food hyperosmotic to their body fluids.

Relation between genetic variants of the ataxia telangiectasia-mutated (ATM) gene, drug resistance, clinical outcome and predisposition to childhood T-lineage acute lymphoblastic leukaemia.

The T-lineage phenotype in children with acute lymphoblastic leukaemia (ALL) is associated with in vitro drug resistance and a higher relapse-risk compared to a precursor B phenotype. Our study was aimed to investigate whether mutations in the ATM gene occur in childhood T-lineage acute lymphoblastic leukaemia (T-ALL) that are linked to drug resistance and clinical outcome. In all, 20 different single nucleotide substitutions were found in 16 exons of ATM in 62/103 (60%) T-ALL children and 51/99 (52%, P = 0.21) controls. Besides the well-known polymorphism D1853N, five other alterations (S707P, F858L, P1054R, L1472W, Y1475C) in the coding part of ATM were found. These five coding alterations seem to occur more frequently in T-ALL (13%) than controls (5%, P = 0.06), but did not associate with altered expression levels of ATM or in vitro resistance to daunorubicin. However, T-ALL patients carrying these five coding alterations presented with a higher white blood cell count at diagnosis (P = 0.05) and show an increased relapse-risk (5-year probability of disease-free survival (pDFS) = 48%) compared to patients with other alterations or wild-type ATM (5-year pDFS = 76%, P = 0.05). The association between five coding ATM alterations in T-ALL, their germline presence, white blood cell count and unfavourable outcome may point to a role for ATM in the development of T-ALL in these children.

Enhanced expression and activity of DNA polymerase beta in human ovarian tumor cells: impact on sensitivity towards antitumor agents.

DNA polymerase beta, one of the most inaccurate DNA synthesizing enzymes, has been shown to confer genetic instability when up-regulated in cells, a situation found in several human cancers. Here, we demonstrated that enhanced activity and expression of this enzyme occur in the human ovarian tumor 2008/C13*5.25 cells, which are resistant to the antitumor agent cisplatin and hypersensitive to 6-thioguanine. We found that translesion synthesis across platinated DNA crosslinks as well as increased incorporation into DNA of 6-thioguanine took place in the 2008/C13*5.25 cells compared to the parental 2008 cells. Such features being molecular signatures of DNA polymerase beta, these findings suggest that deregulation of its expression in cancer cells may contribute to the modulation of the response to antitumor treatments and therefore to tumor progression.

The impact of host plant on the abundance and function of symbiotic bacteria in an aphid.

The black-bean aphid Aphis fabae bears populations of coccoid symbiotic bacteria Buchnera spp. at 2.0-3.2 x 10(7)cells mg(-1)aphid mass and rod-shaped secondary symbionts of uncertain taxonomic affiliation at 0.1-0.6 x 10(7)cells mg(-1)aphid mass. Buchnera provides essential amino acids, supplementing the poor supply in the aphid diet of plant phloem sap. Comparison of the performance of A. fabae containing and experimentally deprived of their bacteria showed that the bacteria caused increased larval mass of aphids reared on Chenopodium album and Papaver dubium plants, but not when reared on Lamium purpureum. In the aphids reared on L. purpureum, the density of the bacteria, especially the secondary symbionts, was significantly elevated, and bacterial-mediated production of the essential amino acid threonine was reduced, even though the essential amino acid content of phloem exudates from L. purpureum had a low threonine content. It is proposed that the shortfall in threonine, possibly compounded by the high density of secondary symbionts, may contribute to the poor performance of the aphids on L. purpureum. This study offers the first evidence to suggest plant-mediated interference with the nutritional function of symbiotic bacteria in any phytophagous insect.

The use of denaturing high-pressure liquid chromatography for the detection of mutations in thiopurine methyltransferase.

The level of expression of the enzyme thiopurine methyltransferase (TPMT) is an important determinant of the metabolism of drugs used both in the treatment of acute leukaemia (6-mercaptopurine and 6-thioguanine) and as an immunosuppressant in patients with autoimmune diseases or following organ transplantation (azathioprine). Studies of enzyme activity in red blood cells have shown that TPMT expression displays genetic polymorphism with 11% of individuals having intermediate and one in 300 undetectable levels. Patients with biallelic mutations and undetectable enzyme activity suffer life-threatening myelosuppression when treated with conventional doses of these drugs. Patients with intermediate activity have an increased risk of drug-associated toxicity. In the Caucasian populations studied to date, intermediate activity is associated with mutations at two sites of the TPMT gene, G460A and A719G (designated TPMT*3A), in 80% of cases. Detection of these mutations has, to date, been based on the analysis of restriction digests of PCR products. In order to simplify this process we have investigated the ability of denaturing high pressure liquid chromatography (DHPLC) to detect the A719G mutation. DHPLC of PCR products from 15 known heterozygotes (TPMT*3A/TPMT*1) and 18 known homozygotes (TPMT*1/TPMT*1) gave a clear pattern difference between the groups and 100% concordance with the results of restriction digests. These results suggest DHPLC represents a valuable technique for accurate and rapid detection of pharmacologically important mutations in the TPMT gene.

Quantifying nutrient production by the microbial symbionts in an aphid.

The symbiotic bacteria Buchnera sp. provide aphids with essential amino acids, nutrients in short supply in the aphid diet of plant phloem sap. The contribution of Buchnera-derived amino acids to net protein growth of the aphid Aphis fabae was quantified from the protein growth of aphids reared on chemically defined diets lacking individual amino acids. The amino acid production rates varied among the nine essential amino acids over the range 8-156 pmol microg(-1)protein day(-1) (for tryptophan and leucine, respectively), equivalent to 0.02-0.33 fmol Buchnera(-1)day(-1). In a complementary metabolic analysis, the aphids incorporated radioactivity from dietary [(14)C]glutamic acid into the essential amino acids isoleucine, lysine and threonine. Incorporation into isoleucine was significantly elevated by the omission of dietary isoleucine, indicating that dietary supply may affect the biosynthetic rates of certain amino acids by Buchnera. Aphids experimentally deprived of Buchnera did not synthesize essential amino acids from dietary glutamic acid. The mortality of aposymbionts was high over 7 days on the phenylalanine-free diet, and their assimilation of dietary leucine was depressed on the complete diet, suggesting that both the absence of bacteria-derived amino acids and the low rates of assimilation of certain dietary amino acids may contribute to the poor growth of these insects.

A comparison of molecular and enzyme-based assays for the detection of thiopurine methyltransferase mutations.

S-Methylation by thiopurine methyltransferase (TPMT) is an important route of metabolism for the thiopurine drugs. About one in 300 individuals are homozygous for a TPMT mutation associated with very low enzyme activity and severe myelosuppression if treated with standard doses of drug. To validate the use of molecular genetic techniques for the detection of TPMT deficiency, we have determined red blood cell TPMT activity in 240 adult blood donors and 55 normal children. Genotype was determined by restriction fragment length analysis of polymerase chain reaction products in a cohort of 79 of the blood donors and five cases of azathioprine-induced myelosupression, and this confirmed a close relationship between genotype and phenotype. In 17 of the 24 cases in which mutations were found, DNA was also available from remission bone marrow. In one of these cases, DNA from the remission marrow sample indicated the presence of a non-mutated allele that had not been seen in the blast DNA sample obtained at presentation. These results indicate that polymerase chain reaction-based assays give reliable and robust results for the detection of TPMT deficiency, but that caution should be exercised in relying exclusively on DNA obtained from lymphoblasts in childhood leukaemia.