The invention provides molecules that target cancer-specific transcription complexes (CSTC), compositions and kits comprising CSTC-targeting molecules, and methods of using CSTC-targeting molecules for the treatment) detection and monitoring of cancer.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention identifies cancer specific transcriptional complexes (CSTCs) that contain isoforms of individual cofactors in melanoma cells. The melanoma specific isoform related transcriptional complexes (TFCs) have altered function compared to wild type TFCs and are part of the molecular machinery that is responsible for malignant transformation. Therefore, melanoma specific TFCs represent attractive drug targets for treatment of melanoma. In addition, these specific TFCs can be used as diagnostic and prognostic biomarkers. Since individual melanomas express different sets of cofactors and TFCs, the efficacy of many current and novel drugs likely depend on composition of TFCs. Modified TECs provide tools for theranostics, i.e., to select patients who will have favorable response to specific treatments. Moreover, the cancer-specific isoforms of transcriptional co-regulators described herein are expressed in a variety of other cancers, extending the usefulness of the disclosed molecules and methods beyond melanoma.

The invention provides molecules that target cancer-specific transcription complexes (CSTCs) compositions and kits comprising CSTC-targeting molecules, and methods of using CSTC-targeting molecules for the treatment and detection of cancer. In one embodiment the invention provides an expression vector comprising a nucleic acid molecule that encodes a CSTC-targeting molecule operably linked to an expression control sequence. In another embodiment, the invention provides an oligonucleotide that encodes a CGSTC-targeting molecule. The nucleic acid molecule may encode the CSTC-targeting molecule in a sense or anti-sense orientation, depending on the intended use. Also provided are host cells containing such expression vectors, which can be used for the production of CSTC-targeting molecules. In some embodiments, the nucleic acid molecule is labeled with a detectable marker, or provided in a composition with a pharmaceutically acceptable carrier.

The invention additionally provides CSTC-targeting peptides and small molecules, including peptides that target transcription complexes modified by cancer-specific isoforms of transcriptional co-regulators. More specifically, the CSTC-targeting molecules of the invention include molecules that modulate the activity of a cancer-specific mediator complex, containing MED24/TRAP100 and isoforms thereof, and a cancer-specific chromatin modifying complex, containing BAF57 and isoforms thereof. The CSTC-targeting molecule may be provided in a variety of forms, as appropriate for a particular use, including, for example, in a soluble form, immobilized on a substrate, or in combination with a pharmaceutically acceptable carrier. In some embodiments, the CSTC-targeting molecule is labeled with a detectable marker, or provided in a composition with a pharmaceutically acceptable carrier.

The methods provided by the invention include a method for inhibiting proliferation of cancer cells comprising contacting a cancer cell with a CSTC-targeting molecule of the invention. Typically, the molecule comprises a peptide, oligonucleotide (e.g. siRNA) or small molecule that modulates the activity of a cancer-specific mediator complex containing MED24/TRAP100 and its isoforms, and a cancer-specific chromatin modifying complex containing BAF57 and its isoforms. In one embodiment, the peptide comprises the amino acid sequence PQMQQNVFQYPGAGMVPQGEANF (SEQ ID NO: 1) or NDRLSDGDSKYSQTSHKLVQLL (SEQ ID NO: 2), that interfere with the function of cancer-specific isoforms of TRAP100 and BAF57, respectively. In a typical embodiment, the peptide further comprises additional sequence selected to facilitate delivery into cells and into nuclei. For example, a cell penetrating peptide (CPP) can be added, such as the following amino acid sequence: RRRRRRR (SEQ ID NO: 3). An example of a peptide that facilitates nuclear delivery is the nucleus localizing signal (NLS) having the amino acid sequence PKKRKV (SEQ ID NO: 4). A peptide of the invention is exemplified by the peptide having the amino acid sequence of PKKRKVRRRRRRRPQMQQNVFQYPGAGMVPQGEANF (TRAP100 P05, SEQ ID NO: 5) or PKKRKVRRRRRRRNDRLSDGDSKYSQTSHKLVQLL (BAF57 P12; SEQ ID NO. 6).

Other methods provided include a method for treating cancer in a subject by administering to the subject a CSTC-targeting molecule of the invention, a method of inhibiting tumor growth, a method for detecting cancer, and a method for inducing apoptosis. The method for inhibiting tumor growth and the method for inducing apoptosis, comprises contacting a tumor or cancer cell with a CSTC-targeting molecule. The method for detecting cancer comprises contacting a tissue specimen with a detectable molecule that specifically binds a CSTC and detecting binding of the detectable molecule. Binding of the detectable molecule is indicative of cancer. Examples of a detectable molecule include a peptide antibody or other molecule that specifically binds to a CSTC. Typically, the cancer is melanoma.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of cancer-specific transcription complexes (CSTCs) that contain isoforms of transcriptional co-regulators specific to human cancers. These molecules provide novel targets for treatment and detection of cancer. Moreover, the data described herein show that molecules directed against the CSTC of the invention are effective in inhibiting proliferation of cancer cells, inducing apoptosis and inhibiting tumor growth. This invention thus provides CSTC-targeting molecules as diagnostic and therapeutic agents for the detection, monitoring and treatment of various cancers.

Transcriptional Complexes as Novel Promising Drug Targets

Transcriptional regulators determine regulatory networks that control gene-specific transcription. The misregulation of these networks is correlated with a growing number of human diseases that are characterized by altered gene expression patterns. This has spurred intense efforts toward the development of artificial transcriptional regulators and/or molecules that modify TFCs to correct and restore "normal" expression of affected genes. Numerous research groups and companies are focusing on development of treatment strategies that target signaling systems mostly kinases and phosphatases, and cell surface molecules that control gene expression and regulate cell division and differentiation. All potential treatments that target signaling and cell surface molecules have one critical problem--cell type specificity. To be effective with minimal side effects, treatments have to affect only diseased cells. Signaling systems and surface molecules are expressed and function in a wide variety of cell populations that makes achieving localized/restricted effects extremely difficult.

It is well known that transcriptional control of individual genes is cell type specific and that different transcription factor complexes are responsible for this specificity. We propose to use the cell type specificity of TFCs to control expression of proteins that are critical for cancer development. Achieving this goal will allow us to manipulate growth and apoptosis of cancer cells. For a long time TFs have been considered to be difficult targets for effective drug development. Recently numerous reports show that small molecules can be developed that interact with specific TFs and control activity of specific TFCs.

Peptide Drugs--Targeting Transcription Complexes

The ultimate action of TFs on target genes, after site-specific DNA binding, is to enhance the recruitment and/or function of the general transcription machinery (RNA polymerase II and general transcription factors TFII-A, -B, -D, -E, -F, and -H; Roeder 1996) on cognate core promoter elements. Recent studies have implicated a large multisubunit coactivator complex, a mediator, as the main pathway for direct communication between DNA binding TFs and the general transcription machinery (reviewed in Malik and Roeder, 2000). Large number of protein/protein interactions determines specificity and function of mediator complex. Peptides that represent interaction surfaces of different transcription factors have been designed and used to manipulate expression of target genes (Kalinichenko et al., 2004, Chinmay et al., 2005, Gail et al., 2005) and control disease.

Prediction of the structures of multi molecular complexes has largely not been addressed, probably due to the magnitude of the combinatorial complexity of the problem. Docking applications have traditionally been used to predict pairwise interactions between molecules. Several algorithms that extend the application of docking to multimolecular assemblies have been developed. We apply these algorithms to predict quaternary structures of both oligomers and multi-protein complexes. These algorithms have predicted well a near-native arrangement of the subunits of mediator complexes. We have used these computational tools to design a small library of peptides that interact with a cancer specific mediator complex and a cancer specific chromatin modifying complex containing cancer specific isoforms of MED24/TRAP100 and BAF57 respectively. Screening of these libraries has identified peptides that affect growth and apoptosis of melanoma cells.

Another critical issue is delivery of therapeutic peptides to cell nucleus where transcription factor complexes are localized and where they perform their function. Cell membranes act as protective walls to exclude peptides that are not actively imported by living cells in order to overcome this barrier for effective delivery of membrane-impermeable peptides, several chemical and physical methods have been developed including electroporation and cationic lipids/liposomes. These methods have been shown to be effective for delivering hydrophobic macromolecules. The drawbacks of these harsh methods are, primarily, the unwanted cellular effects exerted by them, and, secondly, their limitation to in vitro applications. The last decade's discovery of cell-penetrating peptides (CPP) translocating themselves across cell membranes of various cell lines, along with a cargo 100-fold their own size, via a seemingly energy independent process, opens up the possibility for efficient delivery of proteins, peptides and small molecules into cells both in vitro and in vivo. The only consistently found feature present in all CPPs is the high content of basic amino acids, resulting in a positive net charge. Rothbard et al. (2000) showed that cyclosporin A was efficiently delivered into dermal T lymphocytes and inhibited inflammation by linking to a hepta-arginine segment suggesting that positive charge is the required feature for cellular translocation. CPPs possess an appealing set of desirable features for cellular targeting, such as effective delivery in vivo, targeting of the nucleus, applicability to all cell types, no apparent size constraint of cargo and seemingly no immunogenic, antigenic or inflammatory properties.

As delivery vectors, cell-penetrating peptides definitely have proven their value. Their ability to effectively deliver hydrophobic macromolecules into practically all types of cells in vitro, as well as in vivo, without marked levels of cytotoxicity, is impressive.

Combining CPP and TFC interfering peptides opens a new and more effective approach to the targeting of transcriptional complexes with therapeutic peptides.

Cancer and Transcriptional Control

Cancer is a disease of enormous complexity. To date, thousands of genes representing virtually every sub-group of genes have been implicated in cancer. Currently, cancer is thought to develop from proliferating stem or progenitor cells with either mutated genes or rearranged chromosomes. As a result of these genetic alterations tumor cells also possess an altered gene and protein expression compared with non-malignant cells. Whole-genome analysis of gene expression clearly shows specific differences between normal and cancerous cells as well as between cancer types. This suggests that regulatory networks determining the expression of specific genes are different in malignant and non-malignant cells.

Cancer patients have a highly variable clinical course and outcome. Intrinsic genetic heterogeneity of the primary tumor has been suggested to play a role in this variability and may explain it in part (Chang, et al. 2003). Pathological and clinical factors are insufficient to capture the complex cascade of events that drive the clinical behavior of tumors. Extensive analyses of gene expression patterns of a variety of tumors have resulted in an understanding that histologically similar tumors have different gene expression patterns. Oligonucleotide and cDNA microarray techniques have identified molecular subgroups of specific types of cancer (Perou et al., 2000, Hedenfalk et al., 2001, West et al., 2001, Zajchowski et al., 2001). Molecular profiling of tumors has also been used to predict survival of patients and to select patients for adjuvant therapy (van't Veer et al., 2002, van de Vijever et al., 2002).

Cancer Specific TFCs--Novel Drug targets with High Specificity

Well-known characteristics of cancer cells are mutations in variety of regulatory molecules including transcription factors, misexpression of transcription factors, expression of mRNA splice variants encoding specific isoforms of proteins and presence of posttranslational modifications that are not present in normal cells. Mutations and expression of fusion proteins are described in almost every single type of cancer (Leroy H, Roumier C, Huyghe P, Biggio V, Fenaux P, Preudhomme C., CEBPA point mutations in hematological malignancies. Leukemia. 2005 March; 19(3), 329-34, Xia and Barr, Chromosome translocations in sarcomas and the emergence of oncogenic transcription factors. Eur J Cancer. 2005 November; 41(16): 2513-27). Large number of papers report identification of cancer specific or enriched mRNA alternative splice variants. For example, a genome-wide computational screening of 11014 genes using 3,471,822 human expressed sequence tag (EST) sequences identified 26,258 alternatively spliced transcripts/mRNAs of which 845 were significantly associated with cancer (Wang et al., 2003). Several of the gene-specific splice variants have been shown to have a prognostic value. Patients with a high expression of the alternative splice variant of helix-loop-helix transcription factor ARNT have a worse relapse-free and overall survival than patients with a low expression (Qin et al. 2001). As a rule the expression of cancer-specific or enriched alternatively spliced mRNAs is not related to the mutations in splice donor or acceptor sites but due to the changes in the expression of splicing factors.

Our in silico analysis using variety of gene expression and EST databases has revealed a large number of alternative splice variants of transcriptional coactivators including mediator complex that have cell type and diseases specific expression. Not all of these splice variants result in protein isoforms with altered function but represent a cryptic splicing that leads to degradation of mRNAs. However, a number of splice variants become translated into functional proteins that will become part of cancer specific TFCs. These changed TFCs may contribute to the development of cancer. We have generated peptides that affect specifically MED24/TRAP100 and BAF57 isoform containing TFCs and block proliferation and induce apoptosis of melanoma cells.

Therapeutic Approach

Our therapeutic approach is based on identification of cancer specific transcription factor complexes (TFC) that contain mutated and/or altered by posttranslational modifications, and/or alternative splicing, and/or TFC components that are modified by a genomic rearrangement. These cancer specific TFCs have structure and function that are different from structure and function of TFCs in normal, non-cancerous cells.

As an example of our approach, we have specifically identified a number of novel isoforms of transcriptional co-regulators that are components of cancer specific TFCs, including but not limited to mediator complex and chromatin remodeling complex. We have focused on two of these altered complexes: 1. Mediator complex that contains cancer specific isoform of MED24/RAP100. 2. Chromatin modifying complex that contains cancer specific isoform of BAF57.

Using different modeling tools and current understanding of composition, structure and function of mediator and chromatin remodeling complexes we identified potential interactions that are unique in complexes that contain cancer specific isoforms of MED24 and BAF57 and identified potential therapeutic peptides. These peptides interact with a MED and chromatin remodeling complexes and alter the function of transcriptional machinery that results in apoptosis and growth arrest of melanoma cells.

MED24 Isoform Containing Complex

Mediator complex consists of approximately 30 proteins that have different functions and participate in different signaling pathways to respond variety of regulatory signals. MED24 is a part of a MED complex "tail" subunit that is present in specific MED complexes. MED 24 co-precipitates with MED16, MED23 and MED25 that are other subunits of "tail" module. Incorporation of MED24 isoform into "tail" subunit modifies interactions of subunit components and opens opportunity to design interfering molecules that target MED24 isoform specific complex. Therapeutic peptide TRAP100 P05 likely interacts with a "tail" complex structure that is composed of MED16, MED23, MED24 and MED25.

Based on these potential interactions, we have designed a small library of peptides that interact with a cancer specific mediator complex "tail " unit containing cancer specific isoform of MED24/TRAP100. Screening of these libraries has identified a peptide that affects growth and apoptosis of melanoma cells. This peptide does not have a sequence of MED24 isoform and was found to affect transcription via binding to altered structure of "tail" subunit of MED complex.

Chromatin Modifying Complex

Chromatin modifying complex consists of a large number of SWI/SNF/SMARC/BAF proteins, histone acetylases (HAT) and histone deacetylases (HDAC). BAF 57, a specific member of BAF complex and it interacts directly with BAF155, BAF170, steroid hormone receptor co-activators and several HDAC proteins, BAF57 melanoma specific isoform modifies structure and function of a chromatin modifying complex. We have used modeling tools to predict changes in the structure and interactions of chromatin modifying complex containing isoform of BAF571. Based on this information, we have designed a peptide library and screening of this library resulted in the identification of peptides that affect growth and apoptosis of melanoma cells. Specifically, therapeutic peptide which we denoted as BAF57 P12 likely interacts with a chromatin modifying complex subunit that contains BAF155, BAF170 and one or more different HDAC molecules.
 

Claim 1 of 22 Claims

1. An isolated molecule that specifically binds to the isoform-specific portion of SEQ ID NO: 11 or SEQ ID NO: 13, wherein the molecule is a peptide having fewer than 100 amino acids or an antibody.
 

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