(P 277) ANTI-GD2 SYNERGIZES WITH HYPOXIA PATHWAY TARGETING TO BLOCK OSTEOSARCOMA CELL PROLIFERATION AND MIGRATION.

Objective: Osteosarcoma (OTS), the most common primary malignant bone tumor in pediatrics, remains a challenge to treat and cure, especially in case of highly resistant disease. Among the list of extra-tumoral mechanisms enabling the cancer cell resistance, hypoxia and tumor-associated macrophage modulation seem to be in the top ranks. Here, we aim to study hypoxic features in OTS tumors and in innovative in vitro OTS patient-derived models focusing on mTor and HIF-1α as major actors of the hypoxic environment additionally to GD2 overexpression. The ganglioside GD2, known to be a key marker of mesenchymal/osteoblast precursor and a cell surface–associated antigen, is serving as a target of macrophage immunotherapy. So, our objectives were to confirm first the expression of all those biomarkers (e.g., mTor, HIF-1 and GD2) in OTS tumors and models. Thereafter, we evaluate efficacy of rapamycin, irinotecan and dinutuximab beta in single drug approach in each model and proceed to combination assessments to understand their impact on OTS cell proliferation and migration and enable the selection the potential therapeutic combo to use.

Methods: Using in vitro OTS patient-derived models (cell lines and tumoroids), patient-derived xenografts (PDX), tumoroids combining patient-OTS cells and macrophages and tumors’ collection, we, first, determined by CGHarray, immunohistochemistry, immunofluorescence, western blot analysis and quantitative PCR the presence of all hypoxia and GD2 biomarkers. Thereafter, based on proliferation and migration assays, we proceed to drug screening using mTor, HIF1 and GD2 targeting in single or combo approaches. Finally, using PDX, we confirmed the combo approach.

Results: First, we were able to confirm the involvement of mTor, HIF1 and GD2 hyperexpressions in OTS cells and tumors. Using the several models (patiet-derived cell lines, tumoroids and PDX) and combining blockade of those targets, we evidenced in our in vitro OTS models using proliferation assay, western blotting, immunofluorescence, migration experiments a synergism between mTorc1 and HIF-1α inhibitors (e.g., rapamycin and irinotecan, respectively) and when associating anti-GD2 (Dinutuximab Beta). Those data provide the rationale for developing combination therapeutic strategies in resistant OTS. The combo were validated in animal model.

Conclusion: Clearly, we were able to show in patient biopsies, as well as in our patient-derived models that the 3 targets were largely expressed and might be efficiently inhibited to prevent OTS cell proliferation and migration.