There is a significant need for new cancer therapies that enhance efficacy and reduce long-term morbidity. Protein products of tumor-specific chromosomal translocations, which are present only in cancer cells, provide unique targets for anti-tumor therapies1
. These translocations span a broad range of malignancies, including carcinomas, hematopoietic malignancies, and sarcomas2-4
. In many cancers, these translocations lead to novel fusion proteins that both initiate and maintain oncogenesis. While some of these translocations, such as BCR-ABL5
, lead to constitutively activated kinases, the majority lead to fusion proteins that function as transcription factors and lack intrinsic enzymatic activity. These translocation-generated transcription factor fusion proteins are ideal targets of anti-cancer therapies, yet no pharmaceuticals have been developed towards these targets.
The Ewing's sarcoma family of tumors (ESFT) can occur anywhere in the body and most often in the 2nd
decades. ESFT often respond well to initial chemotherapy, yet 40% of patients will develop recurrent disease. The majority of patients with recurrent disease will die from ESFT, while 75 - 80% of patients who present with metastatic ESFT will die within 5 years despite high-dose chemotherapy6
. ESFT contain a well-characterized chromosomal translocation that fuses the amino-half of EWS to the carboxy-half of an ets
-family DNA binding protein7
. The most common fusion protein is the oncogenic transcription factor EWS-FLI1. Elimination of EWS-FLI1 using antisense and siRNA approaches results in the prolonged survival of ESFT xenograft-bearing animals8
, but this approach currently lacks translation to clinical therapy9,10
. Small-molecule targeting would be directed towards the disruption of EWS-FLI1 from established transcriptional complexes, since EWS-FLI1 lacks intrinsic enzymatic activity. The EWS-FLI1 transcriptional complex includes: RNA polymerase II, CREB-binding protein (CBP), and RNA Helicase A (RHA)11-13
. Our previous investigations showed RHA augments EWS-FLI1 modulated oncogenesis, suggesting that this protein-protein complex is particularly important for tumor maintenance13
. Small molecule inhibitors that block RHA interaction by targeting the oncogenic fusion protein EWS-FLI1 would be the first in a new class of anti-tumor therapy.
RHA has a critical role in embryogenesis and thus seems a reasonable partner for an oncogene in a highly undifferentiated tumor. RHA is indispensable for ectoderm survival in gastrulation of mammals14
and is required beyond embryogenesis because RHA null mouse fibroblast cells are not viable (personal communication, Dr. Chee-Gun Lee). However, transient reduction of RHA protein levels in COS cells did not affect the viability15
. RHA provides a transcriptional coactivator role in models of tumorigenesis including NFkappaB16
transcriptomes. RHA binds to DNA in a sequence specific manner upon the promoters of p16INK4a
. The amino-terminal region of RHA is most often the site for protein-protein interactions. CBP binds aa 1-250 of RHA 20
, while additional partners for RHA bind in the amino-terminal region including RNA polymerase II21
. In modulating RNA interference in the RISC complex, RHA and Dicer, TRBP and Ago2 interact in the region of aa 1-272 of RHA22
. EWS-FLI1 binds to RHA in a unique region that is not occupied by other transcriptional nor RNA metabolism proteins13
, thus increasing the attractiveness of this protein-protein target.
Disruption of protein-protein interactions by small molecules is a rapidly evolving field. Proteins with more flexible structures, in some cases disordered proteins, have a greater potential for small molecule binding than rigid proteins because of higher induced fit sampling probabilities23
. A disordered protein is defined, in part, by increased intrinsic movement and the inability to form rigid 3-dimensional structure (for review see24
). EWS-FLI1 is a disordered protein and requires the disorder for maximal transactivation of transcription25,26
. Based on these observations, EWS-FLI1, along with its binding to RHA, may provide a unique drug target.