A Rational Combination Therapy Approach with Radiation Therapy in Ewing Sarcoma

Abstract

Ewing sarcoma (EWS) is the second most common malignant bone tumor primarily occurring in adolescents and young adults. Radiation therapy (RT) is usually used in tumors not amenable to surgery or in those resected with compromised margins. It is also used in metastatic disease. Despite advances in the treatment of EWS that have improved the survival substantially to 70% in localized disease, outcome for metastatic, relapsed and recurrent disease remains dismal. Therefore, newer therapeutic approaches, in combination with conventional chemotherapy and RT are needed to improve outcome. Given known deficits in DNA damage repair in EWS, we hypothesize that adding poly-ADP ribose polymerase inhibitors (PARPis), which selectively target the DNA damage response, to the current treatment combinations may potentially improve the therapeutic outcome. We investigated primary and recurrent standard of care (SOC) chemotherapeutics and PARPis as single agents and in combination with RT in the treatment of several human EWS cell lines using colony forming assay. We also compared the result to a radioresistant osteosarcoma cell line, U2OS. DNA damage was assessed using immunofluorescence microscopy by quantifying γ-H2A.X foci. The effect of drugs on DNA repair was measured using a combined intra-chromosomal homologous recombination (HR) / mutagenic non-homologous end joining (mNHEJ) assay. To determine whether sensitivity of PARPis depends on the expression of PARP1, we established a stable cell line which PARP1 was knocked down when induced with doxycycline. The drug combinations were tested in vivo using orthotopic xenograft models of EWS with image-guided fractionated irradiation and tumor burden was monitored via bioluminescence. At physiologically-relevant concentrations of the PARPi talazoparib (TAL), the combination of RT+TAL showed the highest synergist effect and combined efficacy compared to the other PARPis and SOC chemotherapeutics in human EWS cell lines. Differences in radiosensitivity across systemic agents were partially correlated to their ability to induce DNA damage. Moreover, PARPis did not significantly affect HR or mNHEJ DNA repair pathways and inducible PARP knockdown affected EWS (ES8) cells’ sensitivity to TAL. The combinations of irinotecan (IRN)-TAL-RT was found to have equivalent tumor growth inhibition to IRN-TAL-temozolomide (TMZ), a combination currently under clinical investigation in pediatric patients with recurrent/refractory sarcoma (NCT02392793) in EWS mouse xenografts. Taking together, our work suggested the combination of TAL and IRN may be a useful therapeutic strategy when RT is used in the progressive disease setting.