DDDR-77. Studying drug resistance in GBM preclinical models: generation of TAT Cx43266-283- and TMZ- resistant cells and evaluation of treatment combinations

Glioblastoma (GBM) is the most common malignant primary brain tumor. Despite current standard of care (surgical resection followed by radiotherapy and temozolomide [TMZ] administration), patients diagnosed with glioblastoma have a median survival of 10-15 months. Glioblastoma stem cells (GSCs) can withstand current therapies and self-renew, causing fatal relapses. Our lab designed the cell penetrating peptide TAT-Cx43266-283 (TAT-Cx43), which targets GSCs and increases glioblastoma-bearing mouse survival. Due to the important role of treatment resistance in glioblastoma progression and recurrence, in this study we aim to develop TAT-Cx43- and TMZ-resistant GSCs to evaluate different combination therapies to tackle the disease. In order to study therapeutic resistance, we generated GL261- and SB28-GSC resistant subpopulations by growing them with increasing concentrations of TAT-Cx43 and TMZ. Next, we studied TAT-Cx43 and TMZ effect on the viability of the resistant cells and their control counterparts. Our preliminary observations suggest that TMZ reduced cell viability 144 h after treatment, while we could already see this cell viability reduction 72 h after TAT-Cx43 treatment. However, this anti-tumor effect was greatly reduced on TMZ-resistant and on TAT-Cx43-resistant GSCs, respectively, compared to their parental counterparts. Importantly, no significant cross-resistance was observed, which suggests a potential benefit of a sequential TMZ + TAT-Cx43 combination therapy. Thus, we first determined the TMZ and TAT-Cx43 IC50 of resistant GSCs to choose the most optimal concentrations in vitro and we tested its combination, observing a mild additive effect. In conclusion, exposing GSCs to increasing doses of TAT-Cx43 and TMZ led to the generation of resistant GSCs. These cells show no cross-resistance, suggesting that the sequential administration of the two drugs could contribute to overcome treatment resistance. In fact, our preliminary results in GL261-GSCs further support this observation. Altogether, our results suggest a potential benefit of administering TAT-Cx43 as a combination therapy in a clinical context.