(P 279) PROTEIN PHOSPHATASE 2 A INHIBITION AS A MECHANISM TO ENHANCE DOXORUBICIN ACTIVITY

Objective: Heterogeneity of sarcoma renders challenging the development of new therapeutic strategies. New options are needed, moreover in patients with advanced soft-tissue sarcoma (STS). Doxorubicin (D) is still the standard of first line in advanced STS, and several drug combinations have failed to demonstrate a survival benefit over doxorubicin alone, in phase III trials. Protein Phosphatase 2A (PP2A) is a protein involved in DNA damage repair mechanisms. LB-100, a PP2A inhibitor (PP2Ai), has been described to impair DNA damage repair mechanisms, synergizing with chemotherapy or radiotherapy, especially in cancer cells. We hypothesize that LB100 will synergize with doxorubicin, enhancing its activity and increasing cell death in preclinical models of STS. Our objetives are:
- To determine the potential synergy between LB-100 and doxorubicin in in vitro studies.
- To evaluate the activity of LB-100 plus doxorubicin in patient-derived xenograft (PDX) models of leiomyosarcoma (LMS) and undifferentiated pleomorphic sarcoma (UPS).
- To assess the mechanisms underlying the activity of this combination in both in vitro and in vivo studies.

Methods: STS human cell lines of leiomyosarcoma (AA, CP0024, SK-UT-1 and IEC005) and leiomyosarcoma PDX model, were treated with D, LB100 and D + LB100. Cell viability was assessed by MTS after 72 hours. Based on synergy results, the best treatment sequence was selected for functional studies. Briefly, the effect of D + LB-100 tested in: apoptotic cell death, by western blot (WB) (i.e. cleaved PARP1, cleaved caspase-3) and flow cytometry. DNA damage accumulation, by WB and immunofluorescence (e.g. ATR, ATM, γH2AX, RAD51, RPA foci accumulation). Statistical analysis was evaluated by MTS assay, and IC-50 was determined by fitting a nonlinear regression analysis to each model.

Results: Significant differences between D monotherapy and D + LB-100, based on IC-50 cell viability were found. The increase in apoptosis of all cell lines analysed could be related with PP2A function in the DNA. Two mechanisms were involved, the homologous recombination (HR) and non-homologous end joining (NHEJ). Both repair pathways are active; however, the NHEJ pathway (53BP1...) was generally less efficient than the HR pathway (ATR, ATM, RAD51, RPA yH2AX...) in the absence of a homologous template.

Conclusion: PP2A is involved in the doxorubicin mechanism and LB-100 is a good enhancer of these treatments in STS (at least in leiomyosarcoma). This study opens a new insight to further understand the mechanisms in different sarcoma subtypes, for the translational studies to the clinical patients. A comprehensive analysis of the mechanisms underlying sensitivity or resistance to doxorubicin will generate new research hypothesis for future preclinical and clinical studies.