Throughout my blog posts, I have been looking closely at a paper whose authors claim that KRAS mutations cause acquired resistance to an anti-EGFR therapy drug called cetuximab for colorectal cancers. My last blog post examined the data of one of the cellular models, DiFi, and now I will do the same for the other, Lim1215. These cellular models are used to help define the molecular basis of this secondary resistance, which in the future can help prevent such matters. Lim1215 cells express normal levels of EGFR, while DiFi overexpressed EGFR, but both are similarly sensitive to cetuximab.
Graph (a) depicts the relationship between cetuximab concentration and cell viability, ultimately exhibiting the successful resistance created in the R lines. The overlap of error bars of the R lines show that the resistance is similar between the two, despite method of treatment. Once again, the difference between the R1 and R2 lines isn’t explained. Just like the DiFi R lines, the Lim1215 R1 line was exposed to a constant concentration of cetuximab, while R2 was exposed to a concentration that increased stepwise over time for a year. However, there is a striking difference between the Lim1215 R lines and the DiFi R lines. The Lim1215 R lines were exposed to an overall concentration of 1400 nM over a course of 3 months, while DiFi R lines were exposed to an overall concentration of 350 nM over the course of a year. The specifics and reasons behind this procedure are unclear, making the comparison between the two cell lines questionable. How else are the two cellular models different to require different protocols?
According to the Sanger sequencing results in part (b), which is just a method of DNA sequencing, the two resistant lines show inconsistencies compared to the control, as pointed out by the arrows. The other peaks not pointed out do not match perfectly with the control results, but the peaks seem to be relatively proportional, so they are deemed as insignificant differences. In Lim1215 R1, the difference turned out to be a G12R mutation, which means that at position 12 in KRAS, there is an amino acid substitution from a glycine to an arginine. On the other hand, the difference in Lim1215 R2 is a G13D mutation, where an aspartic substituted a glycine at position 13 in KRAS. Because the mutation differed in R1 and R2, I would assume that the type of cetuximab treatment could possibly affect the type of mutation, but the researchers do not expound further on this topic.
The Western blot analysis in part (c) shows that regardless of cetuximab exposure, active GTP-KRAS is present in the resistant lines but not the parent Lim1215 line. All the other protein expression levels seem similar, emphasizing the significance of the active GTP-KRAS. The evidence of active GTP-KRAS paired with the KRAS mutations magnifies the possibility that there is a correlation between KRAS mutation and acquired cetuximab resistance.
The methods to back up the data in parts (d) and (e) are not focused on much in the paper. Part (d) is a schematic representation of how the G12R and G13D mutations were “knocked-in” to the genome of Lim1215 parental cells. The paper does not explain this procedure or its significance further, but its relationship with the graph in part (e) can help one understand how the authors come to their conclusions. One can assume that the purpose of knocking-in the mutations into the genome of the parent cells is to test if the mutations are the reason why there is resistance, or if they are just side products. Illustrated in graph (e), the control parent line has significantly lower cell viability as the cetuximab concentration increases, consistent with the control in graph (a) as well. The Lim KI G12R, which corresponds to the mutation in Lim1215 R1, has a similar resistance when compared to graph (a). Lim KI G13D, which corresponds to the mutation in Lim1215 R2, is still resistant compared to the control, but does not show the same cell viability compared to R2 in graph (a). Therefore, another variable may play a role in affecting the resistance in R2. However, the mutation in Lim1215 R1 seems to be a very strong candidate.
In the DiFi cells, which I went over in my last blog post, the authors focused on how KRAS amplifications mediate acquired resistance to cetuximab, but with these Lim1215 cells, KRAS mutations also play a huge role. Overall, I found the data to be strong enough to support the claim that KRAS mutations can drive acquired resistance to cetuximab, especially with the knock-in of mutations to form experimental lines to compare to the control parental line. The few concerns I have that aren’t clarified in the paper deal with the differences between the methods of treatment of DiFi and Lim1215 as well as of Lim1215 R1 and Lim1215 R2 to determine if it can explain their difference in resulting mutations. Insight into these may lead to other interesting findings. In addition, I question if any other players can affect resistance and KRAS mutations
From the results of these two cellular models, the authors then needed to determine if KRAS mutation and amplification are clinically relevant. In my next blog, I will dive into the clinical data set.
Sandra Misale, Rona Yaeger, Sebastijan Hobor, Elisa Scala, Manickam Janakiraman, David Liska, Emanuele Valtorta, Roberta Schiavo, Michela Buscarino, Giulia Siravegna, Katia Bencardino, Andrea Cercek, Chin-Tung Chen, Silvio Veronese, Carlo Zanon, Andrea Sartore-Bianchi, Marcello Gambacorta, Margherita Gallicchio, Efsevia Vakiani, Valentina Boscaro, Enzo Medico, Martin Weiser, Salvatore Siena, Federica Di Nicolantonio, David Solit, and Alberto Bardelli. “Emergence of KRAS mutations and acquired resistance to anti EGFR therapy in colorectal cancer.” Nature (2012) 486:7404. Web May 3, 2014.