Anaplastic Lymphoma Kinase: role in specific tumours, and development of small molecule inhibitors for cancer therapy.

The Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase first identified as the product of a gene rearrangement in Anaplastic Large Cell Lymphoma. ALK has subsequently been found to be rearranged, mutated, or amplified in a further series of tumours including neuroblastoma, and Non-Small Cell Lung Cancer. There is strong preclinical evidence that ALK is a driving force for oncogenesis in these cases, and that inhibition of ALK kinase activity results in anti-tumoural efficacy. These observations have sparked the development of small molecule kinase inhibitors, the most advanced of which is currently in clinical testing and which has shown promising preliminary activity in the subset of lung cancer patients whose tumours harbour activated ALK. In this review, we describe the various oncogenic forms of ALK, relevant clinical settings, and give a detailed overview of current drug discovery efforts in the field.

HIRA, a mammalian homologue of Saccharomyces cerevisiae transcriptional co-repressors, interacts with Pax3.

HIRA maps to the DiGeorge/velocardiofacial syndrome critical region (DGCR) at 22q11 (refs 1,2) and encodes a WD40 repeat protein similar to yeast Hir1p and Hir2p. These transcriptional co-repressors regulate cell cycle-dependent histone gene transcription, possibly by remodelling local chromatin structure. We report an interaction between HIRA and the transcription factor Pax3. Pax3 haploinsufficiency results in the mouse splotch and human Waardenburg syndrome (WSI and WSIII) phenotypes. Mice homozygous for Pax3 mutations die in utero with a phenocopy of DGS, or neonatally with neural tube defects. HIRA was also found to interact with core histones. Thus, altered stoichiometry of complexes containing HIRA may be important for the development of structures affected in WS and DGS.

Genomic structure and organization of kringles type 3 to 10 of the apolipoprotein(a) gene in 6q26-27.

Apolipoprotein(a) [apo(a)] is a highly polymorphic glycoprotein covalently linked to the apolipoprotein B-100 of LDL in a particle called lipoprotein(a) [Lp(a)]. High plasma levels of Lp(a) are associated with coronary as well as peripheral atherosclerosis. Plasma levels of Lp(a) show a remarkable variation ranging from 0.1 mg/dl to over 100 mg/dl. The apo(a) gene shows a size polymorphism which resides in the variable number of kringle domains which resemble plasminogen kringle IV. Ten different types of kringle IV repeats have been described, nine of which (kringle IV type 1 and type 3-10) are each supposed to be present in a single copy. The other kringles, namely kringle IV type 2 repeats, vary in number from 3 to 42 between apo(a) alleles and form the basis for the apo(a) size polymorphism. Although an inverse relationship has been observed between the number of kringle type 2 repeats and plasma levels of Lp(a), there are exceptions to this general finding. Indeed, several individuals have been described with similar apo(a) size alleles but very different plasma levels of Lp(a). Genetic studies have linked these differences to the apo(a) locus on 6q26-27, outlining the importance, besides the kringle type 2 repeats, of other regions of the apo(a) gene in contributing to the interindividual differences in the plasma concentration of Lp(a). One of the candidate regions is represented by the non-repeated type-3 to type-10 kringles which are invariably present in each apo(a) allele and whose structural integrity is playing a critical role in the correct assembly of the Lp(a) particle. Biochemical studies with recombinant wild type and mutagenized apo(a) cDNAs with several alterations of the non-repeated kringles have well documented this latter point. As a starting point to search for genetic variations in these kringles associated with different levels of Lp(a), we are presenting the genome organization of type-3 to 10 kringle along with specific PCR primers for easy analysis from genomic DNA. Restriction as well as partial sequencing analyses of the type-3 to 10 kringles region has also provided interesting clues as to the different evolutionary origin of these types of kringle with respect to the polymorphic type-2 kringles.

Short direct repeats at the breakpoints of a novel large deletion in the CFTR gene suggest a likely slipped mispairing mechanism.

In the cystic fibrosis conductance transmembrane regulator (CFTR) gene a few small deletions and only a large, complex, 50-kb deletion have been described so far. We report a second large deletion, which had been hypothesized in a patient affected by cystic fibrosis on the basis of an abnormal pattern of inheritance of the intragenic microsatellites IVS17b/TA and IVS17b/CA. Southern blot analysis revealed the presence of an anomalous band in the patient and her father, in the region encompassing exons 13 - 19, approximately 0.6 kb shorten than the one present in normal controls, in addition to the band of the correct size. Cloning and sequencing the DNA fragments spanning the region of interest demonstrated the presence of a 703-bp deletion causing complete removal of exon 17b in the paternal cystic fibrosis chromosome. This analysis revealed the presence of two short direct repeats flanking the breakpoints. The 3' repeat partially overlapped the IVS17b/CA microsatellite and the number of CA repeated units present in the paternal cystic fibrosis allele was the shortest ever found among chromosomes so far analyzed. These data may suggest that the mechanism for the generation of the deletion may have involved a slipped mispairing during DNA replication, which has not previously been described in the CFTR gene.

Ten allelic apolipoprotein[a] 5' flanking fragments exhibit comparable promoter activities in HepG2 cells.

Plasma levels of the atherogenic lipoprotein[a] represent a quantitative genetic trait that is primarily controlled by the polymorphic apolipoprotein[a] locus on chromosome 6q. The more than 1000-fold variation in lipoprotein[a] plasma levels is explained to a large extent by a remarkable size polymorphism of the apolipoprotein[a] gene which is translated into apolipoprotein[a] isoforms and by unidentified sequence variation in apo[a]. In a recent report, sequence variation in a 1.5 kb fragment from the 5' flanking region of the apolipoprotein[a] gene was associated with different promoter activities, which led to the suggestion that transcriptional control of the apolipoprotein[a] gene might contribute significantly to lipoprotein[a] plasma levels. We have used a reporter gene assay to compare the promoter activities of these 1.5 kb fragments which were cloned from ten well-characterized apolipoprotein[a] alleles. These ten allelic apolipoprotein[a] fragments revealed, despite the same sequence variation as previously reported, comparable and relatively weak promoter activities in HepG2 hepatocarcinoma cells. Promoter activity for the same fragment in non-liver cells and the identification of a liver cell-specific DNaseI hypersensitive site 3 kb upstream from the ATG start codon suggest that longer fragments must be used in order to analyze the transcriptional regulation of the apolipoprotein[a] gene.

A recombination event in the closely linked plasminogen and apolipoprotein(a) gene loci.

Genetic studies as well as in situ hybridisation data have strongly demonstrated that the genes coding for apoprotein(a) and plasminogen are linked and localised to chromosome 6 at band 6q26-27. We describe in this report the presence of a recombination event in a region of approximately 50 kb of DNA separating the two genes. The recombination was found in an Italian family, in which a mutation affecting both plasminogen plasma level and activity of plasminogen activity has been detected. Polymerase chain reaction and sequencing analysis showed the presence of a mutation different from those previously reported in two Japanese families.

Several liver specific DNAse hypersensitive sites are present in the intergenic region separating human plasminogen and apoprotein(A) genes.

The genes coding for plasminogen and apoprotein(a) are clustered, together with two related genes, on the telomeric region of chromosome 6 at band 6q26-27. Moreover, the two genes are 40 Kb apart and transcriptionally pointing to opposite directions. Plasminogen and apoprotein(a) share a high degree of homology in the promoter and 5' flanking regions and their expression is mainly confined to liver. To assess whether common controlling elements mediate the tissue specific expression of these two genes, the region between them was investigated for DNase hypersensitive sites in hepatic cell nuclei.

Molecular characterisation of the human apo(a)-plasminogen gene family clustered on the telomeric region of chromosome 6 (6q26-27).

The genes coding for apo(a) and plasminogen belong to a family of related genes sharing several structural sequences like leader, kringle, and protease domains. YAC cloning has allowed to understand that all these genes are clustered within 400 Kb of genomic DNA on the telomeric region of chromosome 6 (6q26-27). We have now characterized the two remaining members of the apo(a) and plasminogen gene cluster. One of them was found to contain a leader highly homologous to that of apo(a) and plasminogen, followed by several kringle IV-like units, kringle V and protease domains although no tail sequences could be detected. This apo(a)-like gene was found to be expressed at the RNA level in liver although an in-frame stop codon was detected in one of its kringle units. The other member of the cluster besides the leader shows a plasminogen tail-like domain whose sequences contain a frameshift resulting in a stop codon; another mutation, destroying a consensus splicing site, has been found in a large intron separating the exon coding for the leader from the one encoding the tail-like sequences. The structural organisation of this cluster suggests that new arrangements of these four genes will be a likely finding.

Characterization by yeast artificial chromosome cloning of the linked apolipoprotein(a) and plasminogen genes and identification of the apolipoprotein(a) 5' flanking region.

The apoprotein(a) [apo(a)] gene encodes a protein component of the circulating lipoprotein(a) [Lp(a)]. The apo(a) gene is highly homologous to the plasminogen gene. It encodes one of the most polymorphic human proteins, due to variability in the number of repetitions of structures called kringles. In addition, Lp(a) levels vary among individuals by more than two orders of magnitude, the high levels being highly correlated with predisposition to early atherosclerotic disease. To better understand the genetics and function of the apo(a) gene, we have cloned in yeast artificial chromosome vectors DNA fragments comprising the linked apo(a) and plasminogen genes and other members of the plasminogen family. By a combination of pulsed-field gel electrophoresis and genome walking experiments, we have identified the 5' portion and flanking regions of the apo(a) gene.