Ras mutations are amongst the most commonly occurring mutations in human cancer, present in approximately 49% of colorectal and 20% of lung cancers. Of these, mutations in K-Ras G12 and G13 are the most common. Understanding the role of mutant K-Ras in modulating drug response is critical to the successful development of novel therapeutics, and has been hampered by the lack of suitable in vitro tools.
We have generated suites of SW48 and LIM1215 colorectal cancer cells which harbor one of 7 different K-Ras G12 or G13 mutations. This system uses endogenous promoters and enables panels of isogenic cell lines to be studied which differ only by the point mutation of interest, providing patient relevant in vitro model systems.
Assessing how single base changes affect drug responses
The differences in behaviour of the different lines in the K-Ras panel is evident from their differing pathway activation profiles (Figure 1).
To understand the impact of these mutations on drug responses, we profiled the anti-proliferative activity of a panel of over 30 therapeutic agents with clinical relevance in colorectal cancer (Table 1). Strikingly, we found a consistent pattern of resistance towards MEK1/2 inhibitors across the 7 K-Ras variants compared to parental cells (Figure 2A).
Resistance to MEK inhibitors was seen in five chemically distinct classes of compounds. These results were confirmed in a second suite of K-Ras mutant lines generated in LIM1215 cells, where clear resistance to MEK inhibitors was also observed (Figure 2B).
Figure 2. Both SW48 and LIM1215 K-Ras mutant cells show resistance to MEK inhibitors. Proliferation assays were performed using Cell TitreGlo after 6 days drug treatment in SW48 (A) and LIM1215 (B) cell panels. N=3.
While the panel of 25 other therapeutic agents tested displayed a broad range of anti-proliferative activities, we did not identify any consistent differences in response between parental and K-Ras mutant cells (Figure 3).
Figure 3. Anti-proliferative activities of selected therapeutic agents on the SW48 K-Ras mutant suite. Proliferation assays were performed using Cell TitreGlo after 6 days drug treatment. N=2/3.
Specific mutation affects sensitivity to drug combinations
We next investigated the effects of combining anti-cancer agents to understand if the resistance to MEK inhibitors could be overcome by a second targeted agent. We combined the anti-EGFR monoclonal antibody, Cetuximab, with the MEK inhibitor AZD6244.
We used concentrations of Cetuximab that did not produce any anti-proliferative effects alone. We found that concentrations of Cetuximab as low as 0.01µg/ml were able to resensitize K-Ras mutant cells to the effects of AZD6244 (Figure 4).
Figure 4. Cetuximab resensitizes K-Ras mutant cells to AZD6244. The anti-proliferative effect of AZD6244 was assessed in SW48 parental and K-Ras mutant cells in the presence of Cetuximab. Drugs were added in combination for 6 days and anti-proliferative effects assessed by Cell TitreGlo. N=2/3.
These results suggest that upregulation of the EGFR axis may be a potential mechanism in compensating for MEK inhibition. To investigate this, we performed Western blotting on cells treated with a combination of AZD6244 and Cetuximab and assessed the activation of the EGFR and ERK pathways (Figure 5).
Interestingly, treatment with AZD6244 alone did appear to cause a small induction in EGFRY1068 phosphorylation, but this was not inhibited by combining with Cetuximab. However, in the AZD6244-resistant K-Ras G12C and G12D lines, the combined treatment appeared to lead to a greater level of phospho-ERKT202/T204 inhibition than either agent alone. These studies are on-going.
Figure 5. Western blot pathway analysis of SW48 K-Ras cells following treatment with AZD6244 and Cetuximab alone or in combination.
- We have created suites of isogenic K-Ras mutant colorectal cancer cells that provide valuable tools for studying the consequence of K-Ras mutations in a controlled in vitro system.
- We have used this platform to definitively show that K-Ras mutations impart resistance to clinical MEK inhibitors, and that this resistance can be overcome by combining with Cetuximab treatment.
- Our data highlight the advantages of using this system in profiling molecularly targeted agents in order to identify potential mechanisms of resistance, and in rationally identifying combinations.
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|Download the accompanying application note "Pathway activation analysis in X-MAN® SW48 K-Ras Cell Lines: A tool for predicting patient response"||Click here|
|Read about how we've combined KRAS isogenic cell lines, 3D cell culture and CRISPR screening for more definitive target validation||Click here|