Histiocytic sarcoma : generation and utilization of patient derived cell lines and xenograft models to understand tumorigenesis and identify novel treatment approaches

Canine histiocytic sarcoma (HS) is a proliferative malignancy of dendritic and macrophage lineages with rapid progression, and limited response to available treatment protocols. As the disease pathogenesis has been unclear, oncologists rely on a small repertoire of nonspecific strategies for therapeutic interventions. To fill this gap of knowledge, we established clinically relevant tools and model systems of histiocytic sarcoma, and utilized these to ask fundamental questions aimed at identifying novel targets for more effective treatment approaches. We successfully established and fully characterized three HS cell lines derived from neoplasms of dogs from predisposed breeds. These cell lines were utilized for drug screening, including a high throughput screening platform, where potential drug candidates were selected from a pool of about 2,000 compounds. Among the selected drugs, we identified two small molecule inhibitors to be highly effective in vitro at nanomolar concentrations: dasatinib, a multi-tyrosine kinase inhibitor, including members of SRC family kinase, and trametinib, an inhibitor of MEK, from the MAPK signaling pathway. To evaluate the drug efficacy in vivo, we developed an orthotopic xenograft mouse model harboring intrasplenic HS neoplasms. Immunodeficient mice transplanted with canine HS cells into their spleen showed a consistent tumor growth, and presence of metastasis to multiple organs (i.e. liver, pancreas and omentum), recapitulating an aggressive metastatic form of HS, the one in most need for better treatment options. Studies with orthotopic intrasplenic HS xenograft mice treated with either dasatinib or trametinib were conducted with promising results. Both drugs effectively inhibited tumor growth, and most importantly, significantly increased survival time of treated mice. Additionally, oncogenic gain-of-function mutations in PTPN11 gene were identified in the HS cell lines. PTPN11 gene encodes SHP-2, a protein tyrosine phosphatase, engaged in enhancement of signaling downstream of growth factor, cytokine and extracellular receptors, including MAPK and PI3K/AKT pathways. One HS cell line, the BD cell line, carries the PTPN11 E76K mutation; while three cell lines (OD, PJ and DH82) carry the PTPN11 G503V mutation. Moreover, a KRAS Q61H gain-of-function mutation was also found in OD cell line. We found that somatic PTPN11 mutations are common in canine HS, particularly in BMDs, the breed with highest incidence of HS. A study on a large sample of dogs, PTPN11 mutations were present in 43% of HS from BMDs, and were not identified in any of the lymphoma samples, the second most common neoplasm in this breed.We have established important model systems of canine HS through which we were able to identify promising drug candidates for treatment and key signaling pathways that are involved in oncogenesis. Our HS cell lines carry oncogenic drivers that are commonly present in canine HS, and in some human cases of human HS. Our xenograft model has proved to be a good surrogate system for drug efficacy, and lead to the confirmation of two small molecules, dasatinib and trametinib, as warranting further evaluation in clinical trials in dogs with HS, which can also serve as key proof of concept trials for human HS.