Abstract 5021: Assessment of RAS-RAF-MAPK pathway perturbations and PD-L1 expression in an Isogenic 3D tissue-culture model of drug-resistant melanoma

Abstract

Abstract Development of the next generation of anti-cancer drugs, biologics, and immunotherapies is currently hampered by extremely poor success rates of seemingly promising experimental therapies in human clinical trials. This poor success rate is partially due to a lack of biologically relevant cancer model systems. The 2D culture system, while relatively inexpensive and easy to use, may not always be adequately representative of the tumor microenvironment. Conversely, the use of animal models is costly and time consuming. The 3D culture system is able to represent biologically relevant complexities such as cell-cell communication, differential proliferation rates, and compound penetration. Combining 3D culture with CRISPR/Cas9 genome editing technologies can generate powerful cell-based model systems with both physiological relevance and well-controlled genetic and drug-susceptibility profiles. These new models will enable the investigation of specific molecular mechanisms, biofunctional outcomes of newly identified genetic alternations, and targeted therapeutic drug responses within a more biologically intricate context. In this study, we use CRISPR/Cas9 to generate isogenic drug-resistant melanoma models that can be used as either 2D or 3D cancer models, as well as for studies of the acquired drug resistance. Two different models were generated starting with the BRAF-inhibitor sensitive A375 melanoma cell line. We introduced either the NRASQ61K or the KRASG13D point mutations, both of which are known to confer BRAF inhibitor resistance and are commonly encountered in BRAF-resistant tumor samples. We then assessed the susceptibility of these new isogenic lines to BRAF inhibitors in both 2D and 3D model systems. We also determined the specific effect of these point mutations on the RAS-RAF-MAPK signaling pathway, a key component of cell-cycle escape and tumor proliferation. Furthermore, we assessed the impact of these mutations on the expression of Programmed Death-Ligand 1 (PD-L1), which recent advances in cancer immunology have directly linked to cancer immune evasion and poor clinical outcomes. Our results show that A375 melanoma isogenic cells carrying KRASG13D have dramatically increased EGFR expression levels, while isogenic cells carrying NRASQ61K have constant activation of the MEK-ERK pathway. We also found a significant increase in PD-L1 expression in KRASG13D, but not in NRASQ61K A375 cells, indicating that expression of PD-L1 is directly linked to KRAS mutation or the downstream effects thereof, rather than to a general increase in RAS-RAF-MAPK signaling. These results have direct implications for cancer immune checkpoint molecule studies, and highlight the utility of these isogenic melanoma models in both 2D and 3D applications in research and development of novel anti-cancer drugs and combination therapies. Citation Format: Elizabeth Turner, Luping Chen, Johnathan Haag, Alexei Miagkov, Lysa-Anne Volpe, Metewo Selase Enuameh, Robert Newman, Fang Tian. Assessment of RAS-RAF-MAPK pathway perturbations and PD-L1 expression in an Isogenic 3D tissue-culture model of drug-resistant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5021.