RAS Mutation Tropism
, by Siqi Li, David MacAlpine, and Christopher M Counter
Siqi Li is a graduate student in the laboratory of Dr. Chris Counter at Duke University, and is the lead author on the described study regarding the RAS mutation tropism of urethane. David MacAlpine is an Associate Professor in the Department of Pharmacology and Cancer Biology at Duke University. His laboratory focuses on elucidating the mechanisms of genetic and epigenetic inheritance using genomic and computational approaches. Chris Counter is a Professor of Pharmacology and Cancer Biology at Duke University. His laboratory is interested in understanding how oncogenic mutations in RAS genes initiate and promote tumorigenesis.
“Why do we get cancer?” remains one of the most fundamental yet intractable questions in cancer biology. The answer may lie, at least in part, in understanding how the process of tumorigenesis first begins. However trying to capture that one single, ostensibly random mutagenic event, in a single gene, in a single cell, decades before it will manifest as cancer is challenging to say the least.
One entree to study this phenomenon comes from the mutation patterns of RAS genes. As recently discussed on the RAS Dialogue by Dr. Ian Prior from the University of Liverpool, despite there being three RAS genes (KRAS, NRAS, and HRAS), three hotspot mutation positions (G12, G13, or Q61), and six possible substitutions at each position (V,D,C,S,R,A at G12/13 and L,R,K,H,E,P at Q61), amounting to a total of 54 potential oncogenic mutations (even more if one considers non-canonical mutations), the RAS isoform, position, or even the type of substitution of a RAS mutation is often specific to individual cancers. Case in point, KRASG12D/V is the dominant RAS mutation in pancreatic ductal adenocarcinoma while it is NRASQ61R in papillary thyroid carcinoma. As RAS mutations can be initiating, it stands to reason that these mutational ‘tropisms’ reflect the process of tumor initiation that becomes embedded in the genome of the resulting tumors . We thus sought to capture RAS mutation tropism at the time when these mutations are laid down- during tumor initiation. In this regard, the seminal work of Dr. Alan Balmain at UCSF found that mice exposed to the environmental carcinogen urethane primarily develop lesions in one tissue (lung), initiated by one Ras isoform (Kras) with a mutation at one position (Q61) encoding one substitution (L or R depending on the mouse strain) . We envisioned two extreme models to explain this RAS mutation tropism. Either urethane only induces KrasQ61L/R mutations in the lung (e.g. the tropism is a product of the specificity of the mutagen), or urethane induces mutations everywhere, but only a KrasQ61L/R mutation is oncogenic in the lung (e.g. the tropism is a product of biological selection).
To be fair, there is support for both models , so we were agnostic in our approach. We posited that these two models could be differentiated by capturing the mutations urethane induces immediately after carcinogen exposure. The challenge is that the frequency of urethane-induced mutations is extremely low . Here, we took a page from the work of Dr. Evgeny Nudler from NYU, whose laboratory developed the ultra-sensitive Maximum Depth Sequencing (MDS) method to detect rare mutations arising in bacteria populations . We therefore teamed up with Dr. David MacAlpine at Duke University, an expert in DNA technologies, to adapt the MDS assay for the much larger mammalian genome. By sequencing exons 1 and 2 of Kras and/or Hras using this mammalian version of MDS in different tissues of mice one week after urethane exposure and thereafter, graduate student Siqi Li discovered that mutagenic bias accounts for the substitution and position tropism of the carcinogen, in agreement with mutation signatures derived from whole-exome sequencing of urethane-induced tumors by the laboratory of Dr. Alan Balmain , while the Kras locus (transcriptional status and amount/type of protein produced) plays a more prominent role in the isoform and tissue tropism .
In summary, we codify the underlying principles of each level of the RAS mutation tropism of urethane, finding that the tropism of this carcinogen involves elements of both the specificity and selection models. These principles now provide a conceptual framework to not only to inform the mutational bias of RAS genes observed in human cancers, but also to experimentally pick away at questions of, for example, why certain cancers track with specific human populations, the role of the immune system on tumor initiation, and whether external factors like obesity influence the acquisition and expansion of an oncogenic mutation.