WO2008061674A2

WO2008061674A2 - Use of an animal model and method for testing drugs and treatments for cancer in humans

Info

Publication number: WO2008061674A2
Application number: PCT/EP2007/009911
Authority: WO
Inventors: Gerardus Johannes Maria Martens
Original assignee: Synthon B.V.; Van Loo, Karen, Miriam, Johanna
Priority date: 2006-11-24
Filing date: 2007-11-08
Publication date: 2008-05-29
Also published as: WO2008061674A3

Abstract

The present invention relates to the use of a non-human animal in an animal model for studying cancer and/or for assaying novel treatments and drugs for treating cancer in humans, which animal has an alteration in its genome as compared to the wildtype animal, which alteration leads to a modified expression of Aph-1b. The alteration comprises for example the presence of only one copy of the Aph-1b gene as compared to two copies in the wild type genome or the presence of the SNP651 T>G mutation in the said gene.

Description

USE OF AN ANIMAL MODEL AND METHOD FOR TESTING DRUGS

AND TREATMENTS FOR CANCER IN HUMANS

The present invention relates to a non-human animal for use in an animal model for studying cancer in humans, for testing drugs for their suitability for the treatment of cancer in humans and for assaying novel treatments for cancer in humans. The invention further relates to a method for identifying compounds suitable for treating cancer and to animals and an animal model that can be used in the method and to the drugs identified by using the animal model.

Colorectal cancer is a major cause of death in the western world. Current therapies involve surgical resection combined with chemotherapy (cytotoxic drugs and radiotherapy) . The outcome of the disease is highly dependant on an early diagnosis. It is important that persons that are susceptible to the disease are monitored frequently to diagnose the disease in an early stadium. A method to diagnose the susceptibility of individuals to suffer cancer disease is therefore advantageous.

Notch/Wnt is a signalling pathway involved in self- renewal of the intestinal epithelium. Notch signaling is involved in maintenance of undifferentiated state of APC- mutant neoplastic cells. The Wnt cascade is the driving force behind proliferative adenomas and adenocarcinomas of the intestine .

In most tissues, active Notch signaling is associated with an immature cell type. For terminal differentiation, this signaling has to be down-regulated. In some tissues, the opposite is required and Notch signaling is required for differentiation. In the latter tissues, loss of Notch signaling is associated with blocked differentiation and tumour progression.

^• Over-expression of various proteins in the Notch signaling cascade has been found in renal cell carcinoma, prostate cancer, multiple myeloma, Hodgkins and anaplastic lymphomas. Accumulating data indicate that deregulated Notch activity is also involved in the genesis of other cancers

(pancreatic, medulloblastoma, mucoepidermoid carcinoma) . Aphl-b is a multipass transmembrane protein that interacts with presenilin and nicastrin as a functional component of the gamma-secretase complex. The gamma-secretase complex is required for the intramembrane proteolysis of a number of membrane proteins, including the amyloid-beta precursor protein and Notch. Goutte et al., (Proc Natl Acad Sci USA 99(2):775-9

(2002)) concluded that Aphl and presenilins are required for cell surface localization of the Notch component Aph2

(nicastrin) .

Francis et al., (Dev Cell. 3(l):85-97 (2002)) determined that Aphl and Pen2 were required for Glpl/Notch- mediated signaling, both in embryonic patterning and in postembryonic germ line proliferation.

Francis et al. (2002), supra, also observed reduction in gamma-secretase cleavage of beta-APP and Notch substrates and reduction in the levels of processed presenilin. They concluded that APHl and PEN2 are required for Notch pathway signaling .

Nevertheless a direct relation between the Aph-lb protein and cancer has never been established before. It is useful to study mechanisms of cancer and novel drugs and therapies to treat them. Animal models are particularly advantageous for studying the progression and presentation of diseases and offer an opportunity to test the efficacy of potential therapeutics and treatments.

^• In the research that led to the present invention it was surprisingly found that a single-nucleotide polymorphism (SNP) in the human Aph-lb gene is associated with the susceptibility of an individual to develop cancer. It was more in particular found that the SNP in position 651 of the Aph-lb gene which changes the amino acid in position 217 in the Aph-lb subunit of gamma-secretase from F to L, is directly linked with the susceptibility of individuals to develop cancer and particularly colon cancer. Table 1 shows the SNP651 genotype and allele distribution in cancer cases and controls. This finding is then used according to the invention in an animal model. Aph-lb is known as a component of the gamma-secretase complex. Subtle alterations in gamma-secretase subunit composition may lead to a variety of affected developmental signaling pathways and, consequently, aberrations in cell differentiation and growth. SNP651 (T>G) in exon 6 of the Aph-lb gene showed a highly significant difference in the frequency of its genotype and allele between cancer patients and control groups. The genotype frequency was found to be about 6-7% in the group of colon cancer patients against about 3% in controls (Table Ia) .

The genotype frequency for throat cancer was found to be 5.3% (Table Ib) . Table Ia

Aph-lb SNP651 t>g genotype and allele distribution in patients and controls

SNP = Single-Nucleotide Polymorphism NBS = Nijmegen Biomedical Study Table Ib

SNP = Single-Nucleotide Polymorphism

NBS = Nijmegen Biomedical Study

This T>G substitution leads to a change in amino acid residue 217 from phenylalanine to leucine (Phe/Leu) . Amino acid ^"residue 217 is conserved among all known Aph-lb sequences and also among all sequences of its paralogue Aph-

Ia from man to worm (either Phe or Tyr) . In view of the observed magnitude of the association between SNP651 and cancer, the presence of this genotype in an individual is a significant genetic risk factor that together with other genetic or environmental risk factors increases the likelihood of developing one of these disorders or their complications. This is the first finding that in humans a modification in the Aph-lb gene is correlated with a susceptibility to develop cancer.

Since the SNP651 that is associated with the susceptibility of human patients to develop cancer is present in the Aph-lb gene, the inventors contemplated that an animal model that has the same or similar genetic background as a normal or wild type animal and differs only in its Aph-lb gene could be useful for testing drugs and treatments for cancer. Such difference in the Aph-lb gene between control or wild type animal and test animal is detectable by a difference in Aph-lb expression. The modified expression of Aph-lb can be effected in various ways. According to a first embodiment the Aph-lb dosage of the animal gene is altered such that the gamma- secretase activity is either increased or decreased as compared to the wild type expression in animals not having an altered Aph-lb copy number. Alternatively, the animal can harbour a Aph-lb gene that has the SNP651 mutation. This Aph- lb gene is more particularly the human gene. According to a further embodiment the expression of the Aph-lb gene can be altered by means of differences in regulation of gene expression or by other mutations in the gene that affect the Aph-lb expression. According to a first aspect thereof, the invention thus relates to the use of an animal that has an alteration in its "genome as compared to the wild type animal, which leads to a modified expression of Aph-lb, in an animal model for studying cancer and assaying novel treatments and drugs for cancer in humans .

In particular, the alteration leads to a decreased expression of Aph-lb. The alteration in the animal genome may comprise the presence of only one copy of the Aph-lb gene as compared to two copies in the wild type genome. An example of such an animal is the Aphlb I/I rat.

Alternatively, the alteration in the animal genome comprises the presence of the SNP651 T>G mutation in the Aph- lb gene. In this embodiment the Aph-lb gene is preferably a human Aph-lb gene.

The Aph-lb gene is a variant of the human Aph-lb gene as depicted in Fig. 1 (SEQ ID NO:1) that causes the amino acid residue in position 217 of the encoded gamma-secretase component Aph-lb to be an aliphatic amino acid, in particular a leucine. Such a variant Aph-lb gene has for example the nucleotide sequence as depicted in any one of the Figs. 2A-F (SEQ ID NOS:3-8), or a fragment thereof that comprises the codon encoding amino acid residue 217 of the encoded gamma- secretase component Aph-lb or a nucleotide sequence encoding a polypeptide having the amino acid sequence as depicted in Fig. 4 (SEQ ID NO: 9). Alternatively, the variant Aph-lb gene hybridizes under high stringency conditions to a nucleotide sequence selected from the group consisting of the sequences as shown in any one of the Figs. 2A-F (SEQ ID NOS: 2-7) and the complement of the sequence as shown in any one of the Figs. 2A-F (SEQ ID NOS: 2-7), with the proviso that the nucleic acid has a codon selected from TTA, TTG, CTT, CTC, CTA, CTG in the position encoding the amino acid residue in position 217 of the encoded gamma-secretase component Aph-lb.

" According to the first embodiment, two new rat lines were developed via cross-breeding and genetic re-selection as described in Example 1.

These animals (I/I and III/III) are genetically very similar except for the copy number of the Aph-lb gene. Due to the difference in copy number these novel Aph-lb I/I rats are a suitable animal model that is representative for aberrations in the gamma-secretase activity producing cancer in humans. Until the inventors found that in humans the Aph- lb gene is modified in at least a sub-group of patients suffering from cancer, it was not possible to correlate results obtained in Aph-lb I/I rats with predictions in humans.

The invention thus relates to the use of the above described Aph-lb I/I and III/III rats for studying the role of gamma secretase in the development of cancer and for assaying novel treatments for cancer in humans that are based on interfering with the gamma secretase activity.

As used in this application the term Aph-lb I/I rat refers to an animal that has in its genome one copy of the Aph-lb gene but has an otherwise essentially identical or identical genotype as an Aph-lb III/III rat, which has three copies of the Aph-lb gene. The Aph-lb I/I rat is the test animal. The Aph-lb III/III rat can be used as a control animal .

The animal model can be any non-human animal that shows a Aph-lb dosage imbalance or reduced Aph-lb expression in comparison to an animal having the same genotype except for the Aph-lb dosage imbalance. The reduced expression can be the result of a lower gene dosage but also be caused by a modification to the gene such a^'s in a second embodiment of the animal model of the invention harbouring the SNP651 mutation. Such gene dosage imbalance or reduced expression can be induced by crossing and selection, like in the Aph-lb I/I and III/III animals, but also via a transgenic approach.

In a preferred embodiment the model is a result of crossbreeding two rat lines APO-SUS and APO-UNSUS rats as described in Coolen et al., The FASEB Journal express article 10.1096/fj .05-4337fje, published online October 25, 2005. The animal is also referred to as an Aph-lb I/I. The resulting animal is a knockdown of Aph-lb.

According to a further aspect thereof the invention thus relates to the use of an animal model in which the animal harbors the human SNP651 polymorphism in its genome for studying the role of gamma secretase in the development of cancer and for assaying novel treatments for cancer in humans that are based on interfering with the gamma secretase activity. This embodiment of the animal model of the invention can be created in various ways, in particular through knock-out or knock-in techniques.

A knock-out is created by inserting in the endogenous gene a nucleic acid sequence thereby disabling the gene or by replacing the endogenous gene with a gene harboring SNP651. The substitution relies on homologous recombination between the two ends of the disabled gene, which is injected into embryonic stem (ES) cells, and the endogenous gene of the ES cell. The ES cells where a successful recombination event has occurred are selected and microinjected into recently fertilized eggs that are put back into the mouse. The progeny are called chimeras because some of their tissue is derived from the modified ES cell and thus have the disabled gene.

Chimeras are then mated to one another to eventually get a "founder" animal that has the gene knocked-out in all of its cells. Mice have two copies of the Aph-lb gene. By knocking out one of the copies and changing the other copy by transgenic approaches an animal model according to the invention can be obtained. A transgene is a nucleotide sequence which is integrated into the genome of a cell from which a transgenic animal is developed. In the animal model of the invention the transgene has the SNP651 T>G mutation.

Methods for generating transgenic animals, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. No. 4,736,866.

Alternatively, the animal model of the invention can be produced by means of knock-in. Knock-ins replace the mouse Aph-lb target gene with the human variant Aph-lb gene of the invention that has for example the SNP651 T>G mutation or another mutation.

The invention further relates to the use to investigate the role of gamma secretase in the development of cancer and for assaying novel treatments for cancer in humans that are based on interfering with the gamma secretase activity of all animals that have a modification in the Aph- lb gene or in its expression. The invention relates in particular to knockdown animals. Preferred animals are rodents, more in particular the animals are rats.

According to a further aspect thereof, the invention provides a method for identifying compounds suitable for treatment or prophylaxis of cancer in humans, comprising: a) providing a collection of compounds or compositions; b) administering the compounds or compositions of the collection to a non-human animal having an alteration in its genome that leads to a reduced Aph-lb expression level; c) measuring the gamma-secretase activity in the animal before and after administration of the compound; and d) identifying the compounds in the collection that modulate the gamma-secretase activity of the animal. The animal can have all the features as described above.

Figures

Figure 1 shows the 905 bp cDNA sequence of the known human Aph-lb gene (accession AL136671 from GenBank) encoding the variant component Aph-lb of the human gamma-secretase gene as identified according to the invention. SNP651 is the nucleotide at position 651 of the coding sequence in which the ATG corresponds to positions 1-3. Figures 2A-F show examples of variant Aph-lb genes. Figure 3 shows the known amino acid sequence of the human Aph-lb component of γ-secretase as found in the UniProtKB/Swiss-Prot at entry Q8WW43. The length is 257 amino acids, the molecular weight is 28460 Da. In this application the amino acid in position 217 in this figure will always be designated as "the amino acid residue in position 217" thus referring to the protein that naturally occurs in humans, even when the actual position of that amino acid in an amino acid sequence is not position 217. Figure 4 shows the amino acid sequence of an example of a variant Aph-lb protein. List of Sequence ID numbers

SEQ ID^* NOS

Fig^'. 1 SEQ ID N0:l known Aph-lb gene Fig. 2A SEQ ID NO: 2 novel Aph-lb variant

Fig. 2B SEQ ID NO: 3 novel Aph-lb variant

Fig. 2C SEQ ID NO: 4 novel Aph-lb variant

Fig. 2D SEQ ID NO: 5 novel Aph-lb variant

Fig. 2E SEQ ID NO: 6 novel Aph-lb variant Fig. 2F SEQ ID NO: 7 novel Aph-lb variant

Fig. 3 SEQ ID NO: 8 known Aph-lb protein

Fig. 4 SEQ ID NO: 9 novel Aph-lb protein

Fig. 5 SEQ ID NO: 10 SNP651

EXAMPLES EXAMPLE 1

Production of the Aph-lb I/I and III/III animal model of the invention Via cross-breeding and genetic re-selection two new rat lines were developed, one homozygous for the allele with a single Aph-lb gene (the I/I rat line) and one homozygous for the allele with three gene copies (the III/III rat line) .

Initially, systemic administration of apomorphine (1.5 mg/kg sc) was used to select Wistar rats with a high or low susceptibility to this drug (APO-SUS and APO-UNSUS rats, respectively) .

The evoked stereotyped gnawing behavior (APO-SUS:

>500 gnaws in 45 min; APO-UNSUS: <10 gnaws in 45 min) was used to select female and male rats for breeding the two distinct lines (Cools et al. Brain Res. Bull 24, 49-69 (1990)). Phenotyping of the rat lines was reviewed elsewhere

(Ellenbroek & Cools, . Behav. Genet. 32, 349-361 (2002)).

^* For making the animal model of the invention, rats of the^' 21st generation were used to set up a crossbreeding scheme. Four male and four female I/I rats of the APO-SUS line were crossed with four female and four male III/III rats of the APO-UNSUS line, respectively. The offspring (either I/III or III/I) was inter-crossed preventing brother-sister pairing, and the resulting F2 generation was genotyped for the Aph-lb locus by PCR analysis of genomic DNAs.

The rats homozygous for either one or three Aph-lb gene copies were used to generate I/I and III/III lines, respectively. Apart from the Aph-lb locus, these lines have highly similar general genetic backgrounds excluding the contribution of other genetic factors than the Aph-lb dosage imbalance to the phenotype. The crossbred I/I rats showed a significantly higher apomorphine susceptibility than the crossbred III/III rats. Further, the crossbred I/I rats having one single copy of the Aph-lb gene resulted in a diminished expression of Aph-lb protein and in subtle changes in the gamma-secretase activity in multiple tissues.

Claims

1. Use of a non-human animal in an animal model for studying cancer and/or for assaying novel treatments and drugs for treating cancer in humans, which animal has an alteration in its genome as compared to the wild type animal, which alteration leads to a modified expression of Aph-lb

2. Use as claimed in claim 1, wherein the alteration leads to a decreased expression of Aph-lb.

3. Use as claimed in claim 1 or 2, wherein the alteration in the animal genome comprises the presence of only one copy of the Aph-lb gene as compared to two copies in the wild type genome.

4. Use as claimed in claim 3, wherein the animal is an Aphlb I/I animal.

5. Use as claimed in claim 1 or 2, wherein the alteration in the animal genome comprises the presence of the SNP651 T>G mutation in the Aph-lb gene.

6. Use as claimed in claim 5, wherein the Aph-lb gene is a human Aph-lb gene.

7. Use as claimed in claim 6, wherein the Aph-lb gene is a variant of the human Aph-lb gene as depicted in Fig. 1 (SEQ ID N0:l) that causes the amino acid residue in position 217 of the encoded gamma-secretase component Aph-lb to be an aliphatic amino acid, in particular a leucine.

8. Use as claimed in claim 7, wherein the variant Aph-lb gene has the nucleotide sequence as depicted in any one of the Figs. 2A-F (SEQ ID NOS:3-8), or a fragment thereof that comprises the codon encoding amino acid residue 217 of the encoded gamma-secretase component Aph-lb.

9. Use as claimed in claim 7, wherein the variant Aph-lb gene has a nucleotide sequence encoding a polypeptide having the amino acid sequence as depicted in Fig. 4 (SEQ ID

NO: 9) .

^* 10. Use as claimed in claim 7, wherein the variant Aph-lb gene hybridizes under high stringency conditions to a nucleotide sequence selected from the group consisting of the sequences as shown in any one of the Figs. 2A-F (SEQ ID NOS: 2-7) and the complement of the sequence as shown in any one of the Figs. 2A-F (SEQ ID NOS: 2-7), with the proviso that the nucleic acid has a codon selected from TTA, TTG, CTT, CTC, CTA, CTG in the position encoding the amino acid residue in position 217 of the encoded gamma-secretase component Aph- lb.

11. Method for identifying compounds suitable for treatment or prophylaxis of cancer in humans, comprising: a) providing a collection of compounds or compositions; b) administering the compounds or compositions of the collection to a non-human animal having an alteration in its genome that leads to a reduced Aph-lb expression level; c) measuring the gamma-secretase activity in the animal before and after administration of the compound; and d) identifying the compounds in the collection that modulate the gamma-secretase activity of the animal.

12. Method as claimed in claim 11, wherein the alteration leads to a decreased expression of Aph-lb.

13. Method as claimed in claim 11 or 12, wherein the alteration in the animal genome comprises the presence of only one copy of the Aph-lb gene as compared to two copies in the wild type genome.

14. Method as claimed in claim 13, wherein the animal is an Aphlb I/I animal.

15. Method as claimed in claim 11 or 12, wherein the alteration in the animal genome comprises the presence of the SNP651~T>G mutation in the Aph-lb gene.

16. Method as claimed in claim 15, wherein the Aph-lb gene is a human Aph-lb gene.

17. Method as claimed in any one of the claims 11-16, wherein the animal is a rodent, in particular a rat.

18. An animal model for use in the method as claimed in claim 7, which animal model produces a human Aph-lb.

19. The animal model as claimed in claim 18, wherein the Aph-lb that is produced is a variant Aph-lb, the activity of which is altered as compared to the found in a healthy human individual.

20. The animal model as claimed in claim 19, wherein the gamma-secretase activity is decreased as compared to the secretase activity found in a healthy human individual.

21. The animal model as claimed in claims 19 or 20, wherein the phenylalanine at position 217 of the Aph-lb component of the gamma-secretase is replaced by a leucine.

22. The animal model as claimed in claim 21, wherein the Aph-lb component of the gamma-secretase is expressed from a Aph-lb gene harboring the SNP651 polymorphism.

23. Animal model as claimed in claim 22, wherein the Aph-lb component of the gamma-secretase is expressed from a nucleic acid molecule comprising a variant of the human Aph-lb gene as depicted in Fig. 1 (SEQ ID N0:l) that causes the amino acid residue in position 217 of the encoded gamma- secretase component Aph-lb to be an aliphatic amino acid, in particular a leucine.

24. The animal model as claimed in claim 22, wherein the variant Aph-lb gene has the nucleotide sequence as depicted in any one of the Figs. 2A-F (SEQ ID NOS: 3-8), or a fragment thereof that comprises the codon encoding amino acid residue 217 of the encoded gamma-secretase component Aph-lb.

25. The animal model as claimed in claim 22, wherein the variant Aph-lb gene has a nucleotide sequence encoding a polypeptide having the amino acid sequence as depicted in Pig. 4 (SEQ ID NO: 9) .

26. The animal model as claimed in claim 22, wherein the variant Aph-lb gene hybridizes under high stringency conditions to a nucleotide sequence selected from the group consisting of the sequences as shown in any one of the Figs. 2A-P (SEQ ID NOS: 2-7) and the complement of the sequence as shown in any one of the Figs. 2A-F (SEQ ID N0S:2-7), with the proviso that the nucleic acid has a codon selected from TTA, TTG, CTT, CTC, CTA, CTG in the position encoding the amino acid residue in position 217 of the encoded gamma-secretase component Aph-lb.

27. Aph-lb I/I rat.

28. Aph-lb III/III rat.

29. Animal model for studying cancer and/or for assaying novel treatments and drugs for treating cancer in humans, comprising a Aph-lb I/I animal as the test animal and a Aph-lb III/III animal as the control animal.

30. Use of a non-human Aph-lb I/I animal and/or a non-human Aph-lb III/III animal for studying cancer and assaying novel treatments and drugs for cancer in humans.

31. Non-human animal having in its genome a Aph-lb gene having the SNP651 T>G mutation.

32. Non-human animal as claimed in claim 30, which animal is a rodent, in particular a rat.

33. Non-human animal as claimed in claim 30 or 31 for studying cancer and assaying novel treatments and drugs for cancer in humans .

34. Drug for the treatment of cancer in humans, which drug is identified according to the method as claimed in any^¬ one o£ the claims 11-19.