Myc is a transcription factor that binds to a specialized transcription regulation sequence known as an E-box, often resulting in increased gene expression. Deletion of E-boxes can result in decreased gene expression (Greasley, et al. (2000) Nucleic Acids Res. 28:446-453). Myc binds to a target gene by way of one or more E-boxes associated with that gene. However, no single target of Myc seems to account fully for Myc's biological effects, as several Myc targets appear to cooperate to maintain normal physiology, or to create cell transformation when Myc is overexpressed (Levens (2002) Proc. Natl. Acad. Sci. USA 99:5757-75759).
Myc plays a role in regulating cell proliferation, the cell cycle, cell growth, angiogenesis, apoptosis, and oncogenesis. Myc's activity can increase in tumors as a consequence of mutations, chromosomal rearrangements, increased expression, or gene amplification, e.g., see Nesbit, et al. (1999) Oncogene 18:3004-3016; Zeller, et al. (2001) J. Biol. Chem. 276:48285-48291; He, et al. (1998) Science 281:1509-1512; McMahon, et al. (1998) Cell 94:363-374; Erisman, et al. (1985) Mol. Cell Biol. 5:1969-1976; Rochlitz, et al. (1996). Oncology 53:448-454. Elevated Myc activity in cancer cells may be a consequence of mutations in oncogenes other than Myc, e.g., APC or .beta.-catenin (He, et al. (1998) supra). Increased Myc levels have been documented, e.g., in breast cancer and prostate cancer (Liao and Dickson (2000) Endocrine-Related Cancer 7:143-164; Jenkins, et al. (1997) Cancer Res. 57:524-531.
Myc regulates the cell cycle, growth, and apoptosis. Changes in cell cycle regulation can result in increased cell proliferation. When Myc regulates the cell cycle, it can act as a signaling agent that promotes entry of a cell into the cell cycle (Trumpp, et al. (2001) Nature 414:768-773; Holzel, et al. (2001) EMBO Reports 21:1125-1132; Bouchard, et al. (2001) Genes Bevel 15:2042-2047). Myc has been found to act in specific phases of the cell cycle, where certain cell cycle genes, e.g., cyclins and protein kinases, are directly or indirectly regulated by Myc (Oster, et al., supra). The invention provides methods for modulating the cell cycle.
Myc regulates growth, as it plays a role in regulating genes required for protein synthesis, e.g., genes for transcription factors and ribosomal proteins (Greasley, et al. (2000) supra; Zeller, et al. (2001) supra; Menssen and Hermeking (2002) Proc. Natl. Acad. Sci. USA 99:6274-6279). The invention contemplates methods for modulating growth.
Myc regulates apoptosis, Apoptosis can be impaired in cancer cells, as these cells are often, able to avoid removal by cells of the immune system, survive in new locations in the body, or resist chemotherapy (Reed (2002) Apoptosis in The Cancer Handbook (Ed. by M. R. Alison) Nature Publishing Group, London, pp. 119-134). Myc regulates key apoptosis pathway proteins (Nesbit, et al. (1998) Blood 92:1003-1010; Oster, et al. (2002) supra). The contemplated invention provides methods for modulating apoptosis
The (c) MYC gene and two of its relatives, MYCN or MYCL, contribute to the genesis of a wide variety of human tumors. In these tumors, the expression of MYC genes is enhanced, relative to the surrounding or normal tissue, arguing that there is a selective pressure for high expression of Myc proteins during tumor development. For example, the MYCN gene is amplified in a subset of childhood neuroblastoma, correlating with extremely poor prognosis, of the affected, children (Brodeur, et al., (1984) Science, 224, 1121-1124.). In other tumors, expression of a MYC family gene is increased because mutations occur in the signaling pathway that control their expression: one example are the mutations in the APC pathway that affect cMYC expression in colorectal carcinomas (van de Wetering et al., Cell, 111, 241-250 (2002)).
Besides colon cancer, elevated or deregulated expression of c-Myc has been detected in a wide range of human cancers and is often associated with aggressive, poorly differentiated tumors. Such cancers include breast, cervical, small cell lung carcinomas, osteosarcomas, glioblastomas, melanoma and myeloid leukemias (Pelengaris et al., Nat Rev Cancer 2, 764-7 (2002)).
Inhibition of Myc activity is a highly attractive approach for drug discovery in oncology, since recent experimental data suggest that even a brief inhibition of Myc expression may be sufficient to permanently stop tumor growth and induce regression of tumors. Jam et al. Science 297, 102-4 (2002) engineered a conditional transgenic mouse to overexpress Myc, which induced formation of highly malignant osteogenic sarcoma. A brief loss of Myc overexpression caused the tumor cells to differentiate into mature osteocytes that formed histologically normal bone. Felsher and Bishop Mol. Cell 4, 199-207 (1999) showed that transgenic-mice expressing the myc oncogene in hematopoietic cells developed malignant T cell leukemias and acute myeloid leukemias. However, when this gene was switched off the leukemic cells underwent proliferative arrest, differentiation, and apoptosis. Pelengaris et al. Mol. Cell 3, 565-77 (1999) targeted expression of an inducible form of the c-Myc-protein to the epidermis of mice and observed formation of angiogenic premalignant skin lesions, which regressed when the c-Myc protein was deactivated.
In general, specific pharmacological interference with the function of transcription factors has been difficult to achieve. This is particularly true for Myc: despite its obvious value as a potential target for tumor therapy, no drugs have emerged that specifically interfere with its function. For example, screens aimed at disrupting the Myc/Max interface have only yielded compounds with extremely low potency (Berg et al, Proc Natl Acad Sci USA, 99, 3830-3835 (2002)).
Recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elgans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.
Despite significant advances in the field of RNAi and advances in the treatment of pathological processes which can be mediated by down regulating MYC gene expression, there remains a need for agents that can inhibit MYC gene expression and that can treat diseases associated with MYC gene expression such as cancer.
USPTO Backgrounds
USPTO Backgrounds