Assessment of RAS-RAF-MAPK Pathway Perturbations and PD-L1 Expression in an Isogenic 3-D Tissue-Culture Model of Drug-Resistant Melanoma
AACR Annual Meeting 2018
Chicago, Illinois, United StatesApril 14, 2018
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. These poor success rates are partially due to a lack of biologically relevant cancer model systems. The 2-D culture system, while relatively inexpensive to use and easy to analyze, may not always be adequately representative of the tumor microenvironment. Conversely, the use of animal models is costly and time consuming. The 3-D culture system is able to represent biologically relevant complexities such as cell-cell communication, differential proliferation rates, and compound penetration. We use CRISPR/Cas9 genome editing technology to generate carefully designed and precisely edited cell-based 3-D 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 the CRISPR/Cas9 system to generate isogenic drug-resistant melanoma models that can be used as either 2-D or 3-D cancer models, or used in studies designed to further our understanding of the mechanisms of acquired drug resistance. Two different models were generated from the BRAF inhibitor sensitive A375 melanoma cell line. We introduced either the NRAS Q61K or the KRAS G13D 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 cell lines to traditional BRAF inhibitors in both 2-D and 3-D model systems. Using the parental A375 line as a control, 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 KRAS G13D have dramatically increased EGFR expression levels, while isogenic cells carrying NRAS Q61K have constant activation of the MEK-ERK pathway. We also found a significant increase in PD-L1 expression in KRAS G13D, but not in NRAS Q61K 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 2-D and 3-D applications in research and development of novel anti-cancer drugs and combination therapies.
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