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.
References:
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.
