Extracted from the last newsletter from the Paul Nabil Bustany Fund:
Oncogene Addiction in Synovial Sarcoma
by Malay Haldar, MD and Kevin B. Jones, MD
The readership of this newsletter need not be reminded of the critical need for novel therapeutic strategies to improve survival and reduce treatment morbidity for patients with synovial sarcoma.
The Paul Nabil Bustany Fund for Synovial Sarcoma Research has graciously supported the Mario Capecchi Genetics Laboratory at the University of Utah Huntsman Cancer Institute for the last 3 years in efforts to develop genetically engineered mouse models for this lethal disease for the purpose of understanding the underlying molecular mechanisms and to develop and test novel therapeutic strategies.
Translocations are genetic abnormalities caused by rearrangements of parts between different
chromosomes. These often lead to breaking and joining of unrelated genes residing within affected chromosomes resulting in formation of chimerical genes. Synovial sarcomas are associated with a specific translocation between chromosome X and 18 that leads to formation of a signature chimerical gene comprised partly by SYT gene from chromosome 18 and partly by SSX gene from chromosome X. The expression of SYT-SSX chimerical protein is specific to synovial sarcoma tumor cells and is not seen in any other tumor or normal cells of the body. This suggested that SYT-SSX fusion protein may be responsible for the induction of this tumor. Based upon this assumption, we previously developed a genetically engineered mouse model for this rare and lethal human disease by expressing the human SYT-SSX fusion protein in mouse muscle progenitor cells. These mice developed synovial sarcoma like tumors at an early age. The histology and molecular signature of the mouse tumors closely resembled
the human counterpart. Additionally, we discovered that expression of the SYT-SSX fusion protein leads to tumors only when it is expressed within muscle progenitor cells of the engineered mice.
Expression in other tissue type did not lead to tumor formation. This suggested that SYT-SSX fusion protein is an oncogene that induces synovial sarcoma when expressed in “permissive” cell types.
While the aforementioned studies demonstrated that SYT-SSX fusion protein could induce synovial sarcoma, it is not clear whether continued expression of this fusion gene is required for disease progression (as opposed to disease induction). Certain oncogenes are known to be required throughout the lifetime of a tumor cell, a concept known as “oncogene addiction”. If the tumor cellsare dependent or “addicted” to a certain protein for their survival, it makes that protein a valuable therapeutic target. We are currently investigating whether synovial sarcoma cells demonstrate “addiction” to SYT-SSX fusion protein. In our original mouse model, we had the ability to express SYTSSX in any chosen tissue type. However, once turned on, there was no way to modulate subsequent expression of SYT-SSX. To test for oncogene addiction, we needed a new mouse model where we can not only turn on expression of SYT-SSX to induce tumors, but also turn it off to see the effects of the absence of SYT-SSX on tumor progression. To develop such a model, we decided to combine two wellknown tools in genetic engineering: Cre-LoxP based conditional systems and tetracycline-inducible system. The Cre-LoxP system ensures that SYT-SSX is only expressed in specific tissue type and not globally since global expression of SYT-SSX causes embryonic lethality. The tetracycline inducible system allows SYT-SSX expression only in the presence of the antibiotic doxycycline administered via food or water. By combining these two strategies, we developed a mouse model where SYT-SSX expression cannot only be targeted to specific tissue, but can also be modulated at any time via exposure to doxycycline.
Once we had these mice, our first goal was to induce synovial sarcoma by exposing them to
doxycycline. This entails prolonged exposure to various concentration of doxycycline to figure out the optimal doxycycline regimen that will most consistently induce synovial sarcoma. Our results show that tumor induction by doxycycline is highly dependent upon the age of the mouse when such exposure is begun as well as the dosage of doxycycline. We have now successfully optimized the timings and dosing of doxycycline regimen that leads to tumor formation in nearly 100% of the mice.
Analysis of the tumors generated demonstrated high degree of similarity to human synovial sarcomas.
We have now started the final and the most crucial phase of the study—what happens when we turn off SYT-SSX expression in synovial sarcoma? Briefly, our strategy is to divide engineered mice into control groups without doxycycline and an experimental group exposed to doxycycline regimen. Once the experimental group develops tumor, half of them will be taken off doxycycline while the remaining half will continue on doxycycline. The response of the tumor to the presence and absence of doxycycline will be monitored closely by physical examination and imaging at regular intervals. If evidence of tumor stabilization or tumor regression is seen in the absence of SYT-SSX, these tumors will be subjected to rigorous pathological analysis such as histology, immunohistochemistry, expression profiling, etc.
If we observe tumor regression in the absence of SYT-SSX, the importance of the observation goes beyond confirming oncogene addiction. Understanding the molecular mechanisms responsible for tumor regression in the absence of SYT-SSX will provide us with new insights that could be exploited to design more effective targeted therapies. Even if we do not observe tumor regression, this model will allow us to explore combinatorial therapeutic strategies where SYT-SSX expression can be knocked down with concurrent drug therapies to investigate whether they are more effective in combination. There are many more potential uses for such a versatile model and while we wait for our preliminary results, we are excited about the prospects.
Dr. Malay Haldar and Dr. Kevin B. Jones are Co-investigators at the Department of Human Genetics
and Howard Hughes Medical Institute, University of Utah.