An Integrated Chemical Biology Approach Identifies Specific Vulnerability of Ewing's Sarcoma to Combined Inhibition of Aurora Kinases A and B

Abstract

Ewing's sarcoma is a pediatric cancer of the bone that is characterized by the expression of the chimeric transcription factor EWS-FLI1 that confers a highly malignant phenotype and results from the chromosomal translocation t(11;22)(q24;q12). Poor overall survival and pronounced long-term side effects associated with traditional chemotherapy necessitate the development of novel, targeted, therapeutic strategies. We therefore conducted a focused viability screen with 200 small molecule kinase inhibitors in 2 different Ewing's sarcoma cell lines. This resulted in the identification of several potential molecular intervention points. Most notably, tozasertib (VX-680, MK-0457) displayed unique nanomolar efficacy, which extended to other cell lines, but was specific for Ewing's sarcoma. Furthermore, tozasertib showed strong synergies with the chemotherapeutic drugs etoposide and doxorubicin, the current standard agents for Ewing's sarcoma. To identify the relevant targets underlying the specific vulnerability toward tozasertib, we determined its cellular target profile by chemical proteomics. We identified 20 known and unknown serine/threonine and tyrosine protein kinase targets. Additional target deconvolution and functional validation by RNAi showed simultaneous inhibition of Aurora kinases A and B to be responsible for the observed tozasertib sensitivity, thereby revealing a new mechanism for targeting Ewing's sarcoma. We further corroborated our cellular observations with xenograft mouse models. In summary, the multilayered chemical biology approach presented here identified a specific vulnerability of Ewing's sarcoma to concomitant inhibition of Aurora kinases A and B by tozasertib and danusertib, which has the potential to become a new therapeutic option.

Sarcoma Cell Line Screen of Oncology Drugs and Investigational Agents Identifies Patterns Associated with Gene and microRNA Expression

“…Aurora kinase inhibitors are in clinical trials in hematologic malignancies and solid tumors (27) and are in Phase II clinical trials in a broad spectrum of adult soft tissue sarcoma. Preclinically, pediatric sarcoma are sensitive to aurora kinase inhibitors; however, clinical trials have not yet been initiated (28). In pediatric sarcoma xenograft studies, alisertib (NSC759677) had activity in rhabdomyosarcoma xenografts but little activity against Ewing sarcoma xenografts (29); however, higher doses higher than can be achieved in humans were used (30).…”

Targeting protein kinases to reverse multidrug resistance in sarcoma

“…Aurora kinase A and B are overexpressed in various sarcomas, including chondrosarcoma, osteosarcoma, synovial sarcoma, and Ewing sarcoma, and are associated with poor prognosis and metastasis 104–107 . Targeting aurora kinase A and B with small molecule kinase inhibitors, shRNA, and siRNA exhibited inhibition of tumor cells 105, 107 . Moreover, a study demonstrated that the combination of aurora kinase A and B kinase inhibitor VX-680 (Tozasertib) with etoposide and doxorubicin showed synergistic effects in Ewing sarcoma 107 .…”

“…Targeting aurora kinase A and B with small molecule kinase inhibitors, shRNA, and siRNA exhibited inhibition of tumor cells 105, 107 . Moreover, a study demonstrated that the combination of aurora kinase A and B kinase inhibitor VX-680 (Tozasertib) with etoposide and doxorubicin showed synergistic effects in Ewing sarcoma 107 . Recently, another report also confirmed that the combination of VX-680 with cisplatin, doxorubicin, and methotrexate exhibited a synthetic lethal interaction in osteosarcoma cell lines.…”

Combinatorial Drug Screening Identifies Ewing Sarcoma–specific Sensitivities

“…When we checked in detail, however, none of the PKC412 targets we detected by chemoproteomics was found in the mitochondria-related gene expression datasets shown to be altered by doxycycline (42). Prior to LC-MS/MS analysis, drug pull-down samples from both A673 wt and EWS-FLI1 knockdown conditions were verified by immunoblotting for the presence of Aurora kinase A (AURKA), a known target of PKC412 and one of the kinases upregulated by the EWS-FLI1 oncoprotein (43). Although present in the lysates at a moderate level, AURKA was not detected in the pulldown experiment from EWS-FLI1 silenced cells ( Supplementary Fig.…”