WO2009049635A1 - Novel molecules and mechanism for therapy - Google Patents

Abstract

Identified is a sequence in Gli3 partly conserved in Gli2 and not found in Gli1, which contains the Gli repressor domain. This domain, parts of it or variants of it is expressed to achieve gene transcription repression. This will be of particular value for treatment of cancer and other pathologies arising from aberrant Hedgehog signaling or aberrant activation of gene transcription by Gli proteins.

Description

Novel molecules and mechanism for therapy

TECHNICAL FIELD

This invention relates generally to the fields of biology, medicine, oncology and developmental biology. More specifically, the invention relates to the regulation of gene transcription for the treatment of disorders related to Hedgehog signaling and GIi transcription factor activity.

BACKGROUND ART

The Hedgehog proteins are important signaling molecules that control a multitude of cellular processes important for embryonic development and for numerous other morphogenic/developmental processes in multicellular organisms [I]. In vertebrates the pathway controls the activity of the transcription factors of the GIi family. These proteins are of the Zn-finger DNA binding family and the balance between GIi gene repressor and activator forms in part determines the transcriptional outcome. GUI and GH2 are mostly gene activators and GH3 mostly a gene repressor, although both Gli2 and Gli3 can undergo proteolytic degradation to form an N-terminal gene transcription repressor. A very good example of how the balance between these forms determines cell fate is from the developing spinal cord, where several different neurons are formed due to the distance from the Hedgehog source and differences in the ratios of GIi repressor and activator forms [2].

Several mutant variants of Hedgehog pathway genes have been identified and been associated with developmental pathology and cancer [3]. Most notably was the discovery that Gorlin syndrome (also called Nevoid Basal Cell Carcinoma syndrome; NBCCS) was due to mutations in the gene for the Hedgehog receptor protein Patched (Ptch) [4-6]. These patients exhibit developmental defects and all develop basal cell carcinomas of the skin. They are also predisposed to several other cancer types like medulloblastoma, fibromas and rhadomyosarcoma. Since this discovery aberrant Hedgehog signaling has been linked with numerous cancer types [3].

Several studies have focused on the use of Hedgehog pathway inhibitors to suppress tumor growth and metastasis. Cyclopamine, an inhibitor of Smoothened (Smo), which is upstream of GIi proteins and activated by Hedgehog, has been used extensively to suppress the pathway. Cyclopamine has for example been shown to inhibit lymphomas [7], melanomas [8], myelomas [9] and gliomas [10]. Cyclopamine is now in clinical phase I trials for treatment of cancer. Also inhibitors of Gli-mediated transcription have been determined and were shown to be able to inhibit tumor cell growth [11]. In general, inhibition of the Hedgehog pathway and GIi induced transcription has vast clinical potential for treatment of cancer and developmental pathology [3, 17].

It has been shown that in the N-terminal half of GH2 and Gli3 there is significant gene repression activity [12, 13]. We have identified a peptide in GH2 and GH3 that is not present in GUI and which inhibit both basal and GIi induced gene transcription. These parts are termed GH2RD and GH3RD respectively, where RD means repressor domain. This part is distinct from other parts of the GIi proteins that are involved in regulation of transcription. Our data support that this part mediate transcriptional repression by an unknown mechanism and suggest that introducing this part into cells will inhibit transcription generally and in particular GIi induced Hedgehog pathway mediated transcription.

Our discovery is of vast significance since the use of Gli2RD and GH3RD, variants or parts of these, as described in this invention, allow the inhibition of gene transcription, hi particular it will allow the inhibition of transcription induced by the Hedgehog signaling pathway and GIi proteins. Thus, we can target the last step of the pathway circumventing any steps of the pathway above GIi and treat for the aberrant signaling due to any mutations in genes that are inducing GIi expression and activity. This is likely to have a strong impact on cells affected by aberrant Hedgehog singling and/or too high GIi activity as seen in many cancers.

DISCLOSURE OF THE INVENTION

One aspect of the invention is a method of introducing the GH2RD, GH3RD or variants thereof into cells where gene transcription repression is required. By introducing either the polypeptides or the nucleotides that encode these it will be possible to inhibit unwarranted transcription.

Our invention is based on the notion that GH2RD and GH3RD peptides, any smaller part of these and mutant variants of these, as described in this application, can be introduced into normal or pathogenic cells and there inhibit gene transcription. This would be particularly interesting in cancer cells where the Hedgehog pathway plays a significant role. By introducing the peptides or derivatives of them it is possible to change the pattern of gene expression and therefore also the fate of the cancer cells leading to cancer prevention.

The GH2RD and GH3DR peptides as defined below are from the N-terminal section of their respective proteins and can be identified in GH2 and GH3 from all vertebrates. All these sequences are included and termed according the species. All variants, natural or artificial, of the sequences are included.

With basis in our functional analyses of the peptides in cell cultures it is shown that these peptides are gene transcription repressors. Peptides can be introduced by several means into cells. The invention covers all artificial means of expressing or introducing GH2RD and GH3RD or variant or parts of these in cells, including expression of the nucleotides (DNA or RNA) that encode these peptides.

The invention is aimed at suppressing gene transcription in context where this can be of significance for the cell function, in particular for treatment of disorders involving Hedgehog signaling and/or GIi induced transcription such as cancer.

Another aspect of the invention is related to introducing the peptides in cells. Introduction of the peptides in cells can be achieved using for example variants of adenovirus and the DNA sequence for the peptides can introduced together with other coding sequences that produce peptides that will target the GH2RD and GH3RD peptides to specific sites in the cells. This could for instance be together with GaM-DNA binding domain that will bind to DNA sites in the nucleus.

Another aspect of the invention is producing the peptides. The peptides can also be produced in bacterial culture and purified from this. Purified peptides can then be analyzed and used for introduction into cells using a variety of methods, for example by using liposomes as carriers.

The features and advantages described herein are not all-inclusive and, in particular, many additional features and advantages will be apparent to one of ordinary skill in the art in view of the drawings, specification, and claims. Moreover, it should be noted that the language used in the specification has been principally selected for readability and instructional purposed, and not to limit the scope of the inventive subject matter. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates effects of different GLB and GLIl constructs on 12 GLI12-luc reporter activity in HEK 293 cells.

Fig. 2 illustrates that GLD repressor domain is required for repression.

Fig. 3 is a deletion analysis of GLI3 repressor domain

Fig. 4 is an expression analyses of the 6xHis-Gli3RD peptide in E. coli cells by SDS-PAGE.

MODES FOR CARRYING OUT THE INVENTION

The invention is now illustrated by a reference to the enclosed Figs.

It has been shown that in the N-terminal half of both Gli2 and Gli3 are sequences that in different way suppress gene transcription [12,13]. The so-called PHS domain (residues 1-694) of Gli3 is overexpressed in the Pallister-Hall syndrome and is thought to suppress Hedgehog induced gene transcription in these patients [14]. Expression of the GH3-PHS peptide in cell culture has confirmed that it may act as a gene transcription repressor [12].

We have investigated this further and can report that the GH3-PHS peptide suppress GIi 1 induced transcription (example 2, Fig. 1). In order to pinpoint the region in PHS with repressor activity we aligned the sequences of GIi 1, Gli2 and Gli3 and show that in GH2 and Gli3 there are unique sequences that mediate the gene transcription repression. The repressor sequences of human GH2 and Gli3 are shown below (example 1, Fig. 2).

The key experiment has been to show that these sequences possess the gene transcription repression activity. We have made cDNA construct of the common parts of the GH2RD and GH3RD to achieve expression in transfected mammalian cells and analyze the effect on gene transcription. One such important construct was the Gal4DBD-Gli3-105-236 that was shown to reduce transcription 3 times (example 3, Fig. 3).

In other experiments parts of these repressor domains was removed from either the full-length proteins or from the PHS domains. When cDNA constructs of this kind were expressed in cells, the transcriptional repression was lost (example 2). So removing or disrupting the repressor domain of Gli2 and GH3 increases transcription and expression the repressor domain or parts of this will increase repression.

This may have vast impact on the pathologies that follow aberrant activation of the Hedgehog pathway like it is seen in many cancer types. Introducing a GIi repressor domain or a variant thereof can inhibit this transcription and restore the cell to a non-pathological state. In fact one may be able to suppress the transcription of other pathological cells if the mechanism that this repressor domain utilize is a general gene transcription repression mechanism.

The expression of the repressor domains can be achieved easily in cell culture experiments like for the cell analyses described (example 2, Fig. 1 and 2). There are many different transfection reagents and procedures that can bring cDNA constructs to these cells, which then make expression of the constructs and produce the peptides themselves.

In order to bring the cDNAs into pathological cells in vivo it is anticipated that more sophisticated mechanisms are utilized. These include for instance the use of adenoviral particles that can be designed to target the right cells [15].

In order to investigate the peptides and later produce them for therapy, we have initiated expression in the bacterial strain E. coli BL 21 (example 4, Fig. 4). A His-tagged version of the GH3RD (residues 105-236) was expressed in a fed-batch culture and induced by IPTG. The analyses show that a large portion of the peptide is in an insoluble fraction but also that a significant part is in a soluble fraction. His-tagged proteins can be purified with Ni-columns or Ni-beads. We used Ni-beads to bind the His-tagged GU3RD peptide and could purify the peptide to almost complete purity. We have initiated analyses of the structure of the peptide and will work on expression of other variants of this. Later the aim is to produce a highly stable repressor peptide that can be introduced into pathological cells, for instance cancer cells.

EXAMPLES

Example 1

The human GIi repressor domain peptide sequences (single letter code).

The GH3RD peptide human version residues 33 to 246 of GLI3 - SEQ ID NO. 1 AVASSTTSNEDESPGQTYHRERRNAITMQPQNVQGLSKVSEEPSTSSDERASLIKKEIH GSLPHVAEPSVPYRGTVFAMDPRNGYMEPHYHPPHLFPAFHPPVPIDARHHEGRYHY DPSPIPPLHVPSALSSSPTYPDLPFIRISPHRNPTAASESPFSPPHP YINP YMD YIRSLHCSP SLSMISAARGLSPTDAPHAGVSPAEYYHQMALLTG

The GH3RD peptide human version residueslO5 to 236 of GLI3 (investigated) - SEQ ID NO.

2

GTVFAMDPRNGYMEPHYHPPHLFPAFHPPVPIDARHHEGRYHYDPSPIPPLHVPSALS SSPTYPDLPFIRISPHRNPTAASESPFSPPHP YINP YMD YIRSLHCSPSLSMISAARGLSPT DAPHAGVSPAE

The GH2RD peptide human version residues 32 to 185 of GLI2 - SEQ ID NO. 3.

ASPLVVAAAAAAAVAAQGGVPQHLLPPFHAPLPIDMRHQEGRYHYEPHSVHGVHGP

PALSGSPVISDISLIRLSPHPAGPGESPFNAPHPYVNPHMEhYLRSVHSSPTLSMISAARG

LSPADVAQEHLKERGLFGLPAPG

These sequences and the corresponding sequences in all vertebrate GIi proteins are included in the patent. Also mutant derivatives or shorter versions of the peptides, as well as corresponding invertebrate peptides are included. The patent covers any use of these peptides in cellular context for regulation of gene transcription.

Example 2

Analyses of the N-terminal PHS domain of Gli3.

We used a GIi reporter construct and different sets of GIi cDNA vectors to analyze the gene transcription activation and repression in human 293 cells as described [15]. GIi 1 strongly induces transcription but GH3 and GH3-PHS domains inhibit this induction. Particularly the GH3-PHS is a strong gene transcription repressor.

Fig. 1 illustrates these analyses.

We then made a similar set of analyses in the NIH3T3 clone Shh-LIGHT2 cells [16]. This cell line has a similar reporter incorporated into the genome and therefore the repression analyses are now in a more in vivo-like situation. Also in these cells GIi 1 induces transcription and GH3 and GH3RD represses this. Furthermore, when a large part of the repressor sequences are removed from the Gli3 or the GH3RD constructs this repression is lost. So this part is crucial for repression.

Fig. 2 illustrates these analyses.

Example 3

Expression of a large section of GH3RD with gene transcription repression activity.

We have expressed the part of the GH3RD that is common also to GH2 in a construct with the Gal4-DNA binding domain (Gal4DBD-GH3 RD(106-226)) and show that this construct will inhibit basal transcription in 293 cells. Also shorter versions have some repressor activity and it is suggested that there are two major parts that have repressor function. The constructs are shortened from the C-terminal so deletion 7 is the first and have the highest repression activity.

Fig. 3 illustrates these analyses.

Example 4

Expression and purification of a recombinant GH3RD protein.

The recombinant GH3RD (residues 105-236) peptide was expressed in E. coli BL 21. The fermentations were carried out in fed-batch regime with the aim to achieve biomass densities above 20-3Og wet cell paste per liter. The M9 minimal medium was applied and the solution of glycerol was used as the carbon and energy source. The addition of the glycerol solution was carried out under computer control in a way that its concentration was maintained low in the fermentor to avoid the inhibition by metabolic by-products that are synthesized at high glycerol concentrations. The culture was grown at 25°C to optical density (OD) about 5-6 units after which the temperature was decreased to 20°C. Adding IPTG to final concentration of 0.3 mM then induced the synthesis of the recombinant protein. 10 h after the addition of the inducer when the OD had reached to about 12 units the culture was harvested by centrifugation 20 min. at 8000g and the cell pellets frozen at -70°C until use. Fig. 4 illustrates these analyses.

The exemplary embodiments presented herein illustrate the principles of the invention and are not intended to be exhaustive or to limit the invention to the form disclosed; it is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

REFERENCES

1. Hooper and Scott (2005) Communicating with Hedgehogs. Nat. Rev. MoI. Cell Biol. 6, 306-317.

2. Fuccillo et al. (2006) Morphogen to mitogen: the multiple roles of hedgehog signaling invertebrate neural development. Nat. Rev. Neurosci. 7, 772-783.

3. Ruiz i Altaba et al. (2002) GIi and Hedgehog in cancer: Tumours, embryos and stem cells. Nat. Rev. Cancer 2, 361-371.

4. Hahn et al. (1996) Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 85, 841-851.

5. Johnson et al. (1996) Human homolog of patched, a candidate gene for the basal cell nevus syndrome. Science 272, 1668-1671.

6. Gailani et al. (1996) "The role of the human homologue of Drosophila patched in sporadic basal cell carcinomas." Nature Genet. 14, 78-81.

7. Dierks et al. (2007) Essential role of stromally induced hedgehog signaling in B-cell malignancies. Nat. Medicine 13, 944-951.

8. Stecca et al. (2007) Melanomas require Hedgehog-Gli signaling regulated by interactions between GUI and the Ras-Mek/Akt pathways. Proc. Natl. Acad. Sci. USA 104, 5895-5900.

9. Peacock et al. (2007) Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proc. Natl. Acad. Sci. USA 104, 4048-4053.

10. Clement et al. (2007) Hedgehog-Gli 1 signaling regulates human Glioma growth, cancer stem cell self-renewal, and tumorigenicity. Curr. Biol. 17, 165-172. 11. Lauth et al. (2007) Inhibition of Gli-induced transcription and tumor cell growth by small- molecule antagonists. Proc. Natl. Acad. Sci. USA 104, 8455-8460.

12. Shin et Ia. (1999) GLD mutations in human disorders mimic Drosophila Cubitus interruptus protein functions and localization. Proc. Natl. Acad. Sci. 96, 2880-2884.

13. Sasaki et al. (1999) Regulation of Gli2 and Gli3 activities by an amino-terminal repression domain: implication of Gli2 and GH3 as primary mediators of Shh signaling. Development 126, 3915-3924.

14. Kang et al. (1997) Gli3 frameshift mutations cause autosomal dominant Palliser-Hall Syndrome. Nat. Genet. 15, 266-268.

15. Kreppel and Kochanek (2007) Modification of Adenovirus Gene Transfer Vectors With Synthetic Polymers: A Scientific Review and Technical Guide. MoI. Therapy (in press).

16. US2003/0049246

17. US2007/0009530

18. WO2007/024971

Claims

1. A GU3RD polypeptide as set forth in SEQ ID NO. 1 or SEQ ID NO. 2.

2. A GU2RD polypeptide as set forth in SEQ ID NO. 3.

3. A method for using the polypeptide of claim 1 or 2 into a cell for cellular gene transcription regulation.

4. A nucleotide sequence for encoding the polypeptide of claim 1 or 2.

5. A method for introducing the nucleotide of claim 3 into living cells.