Hayley McDaid Ph.D.

Recipient of the LUNGevity Foundation/Joan’s Legacy Research Grant. Funded equally by Joan's Legacy and the LUNGevity Foundation.

Lay Description

Cancer is caused by the accumulation of somatic mutations and / or chromosomal rearrangements in key proteins that have a central role in cellular homeostasis. Such proteins participate in a tightly regulated signaling network that in normal cells regulates the balance between cell growth and death. Deregulation of one of these networks, RAS-AKT, is causally involved in cancer initiation and propagation in many different tumor types. Lung cancer is a highly heterogeneous disease and somatic mutations have been documented in approximately 500 known human genes. This high degree of genetic variability is a major cause of innate drug resistance in lung cancer. The RAS and AKT components of the network communicate via interactions at numerous levels that involve positive and negative feedback mechanisms. This facilitates the integration of extracellular signals to efficiently regulate cell growth. Importantly, this network converges at the level of protein translation. Protein translation is an extremely complex process that is primarily controlled at the initiation step, in which many proteins that are activated by extracellular stimuli, assemble on mRNA’s to direct their translation into protein. Protein translation regulation is an active area of research in oncology since it has been determined that deregulation of the initiation step of translation is common in many tumor types, including lung cancer.

The role of AKT - mTOR signaling in the regulation of translation initiation is well known, however previous studies by we and others have determined a role for RAS signaling in this process, although most of these studies are emerging and not well-defined. We have determined that lung cell lines derived from patients with adenocarcinoma or bronchioalveolar adenocarcinomas that have mutations in B-RAF (a protein that participates in RAS signaling) have an altered expression profile for a protein that regulates translation initiation.

Furthermore, lung tumors with mutations in B-RAF have preferential sensitivity to MEK-inhibitors. These MEK-dependent mutations of BRAF in lung cancer occur predominately in women and are independent of smoking status. In addition, K-RAS mutations (a protein that also participates in RAS signaling) are found in 29-22% of in adenocarcinomas and bronchioalveolar adenocarcinomas and like B-RAF mutations these may demonstrate sensitivity to MEK-inhibitors. Presently, there are two highly specific MEK-inhibitors currently undergoing clinical evaluation, PD0325901 (Pfizer) and AZD6244 (AstraZeneca). Thus, there is great potential for MEK-directed therapy to be utilized in the treatment of lung cancers that have a defined genotype. This enthusiasm is presently moderated by the fact that the molecular determinants of sensitivity to MEK-inhibition have not been sufficiently characterized in either adenocarcinomas, or bronchioalveolar adenocarcinomas.

Since B-RAF and K-RAS mutations may be preferentially sensitive to MEK-inhibition, the analysis of actively translated RNA species in mutant versus wild-type lung cancer cells after treatment with the MEK-inhibitor PD0325901, may help identify a gene signature that is associated with response to MEK-therapy and potentially may be used to screen relevant patient populations.

A library of B-RAF and K-RAS mutations that have been characterized and known to occur in lung cancer (specifically adenocarcinomas and bronchioalveolar adenocarcinomas), will be made and their sensitivity to the MEK inhibitor PD0325901, relative to the wild type proteins will be evaluated. Subsequently, mRNA species that are actively translated into protein will be identified, and the effect of the MEK-inhibitor, PD0325901, on the expression of these specific mRNA species will also be determined. This expression ‘signature’ will be determined for both B-RAF and K-RAS mutant cell types and may predict response to MEK-directed therapy. This work has the potential to identify specific lung cancer genotypes that will benefit from the new class of MEK-directed therapies and as such, offer a highly selective targeted therapy for the approximate one third of lung cancer patients who tumors have somatic mutations in either K-RAS or B-RAF.

Scientific Abstract

Dependence on a particular abnormality for maintaining a cancer phenotype provides opportunity for therapeutic intervention, as illustrated by the recent finding that tumors with BRAF V600E mutations have preferential sensitivity to MEK-directed therapy. This paradigm also applies to the predominately non-V600 mutations that comprise the 5% of BRAF mutations in lung cancer. These MEK-dependent mutations of BRAF in lung cancer occur predominately in women and are independent of smoking status. Conversely, K-RAS mutations are found in approximately 18% of all lung cancer and are associated with smoking history. The high prevalence of K-RAS mutations in adenocarcinomas and bronchioalveolar adenocarcinomas (approx. 22 – 29%, respectively) warrants the use of targeted therapies specific for this genotype. The same principle applies to lung adenocarcinomas that are B-RAF mutant, although these occur at a much lower frequency, relative to K-RAS mutants. There are two highly specific MEK-inhibitors currently undergoing clinical evaluation, PD0325901 (Pfizer) and AZD6244 (AstraZeneca). Thus, there is great potential for MEK-directed therapy to be utilized in the treatment of lung cancer. This enthusiasm is presently moderated by the fact that the molecular determinants of sensitivity to MEK-inhibition have not been sufficiently characterized in either adenocarcinomas, or bronchioalveolar adenocarcinomas.

The role of MAPK signaling on protein translation is not well-defined. The RAS pathway, via activated ERK, phosphorylates mitogen-activated protein kinase signal-integrating kinases (Mnk1/2) and p90 ribosomal S6 kinase (RSK) that respectively, directly and indirectly regulate the activity of the eukaryotic translation initiation factor, eIF4E. BRAF mutant lung cell lines demonstrate a 4EBP1 phosphorylation profile that is distinct from non-BRAF mutant cell lines, suggesting that oncogenic transformation via mutated B-RAF is associated with altered protein translation, relative to wild type RAF.

Based on preliminary evidence, we hypothesize that lung specific-B-RAF mutations are associated with significantly altered rates of mRNA translation, and thus distinct classes of mRNA species are preferentially translated in B-RAF mutant cell lines, relative to either wild type, or K-RAS mutant cells. Since B-RAF mutations may be preferentially sensitive to MEK-inhibition, the analysis of mRNA translation profiles in B-RAF mutant versus wild-type by gene expression profiling of actively translated mRNA’s, may help identify a gene signature that is associated with response to MEK-therapy and potentially may be used to screen relevant patient populations. Likewise, the same principle may be applied to K-RAS mutant cell lines that demonstrate preferential sensitivity to MEK-inhibitor therapy. Furthermore, we hypothesize that altered expression of S6ribosomal protein at specific resides that are regulated by ERK-RSK signaling, may be prognostic of outcome to MEK-inhibitor therapy, and that this occurs in the presence of STK1 / LKB1 mutations.

The high degree of genetic heterogeneity in lung cancer cell lines impedes accurate pharmacogenomic analyses. Thus, to circumvent this problem, a library of lung specific mutants of B-RAF, K-RAS and STK11 will be stably expressed in HEK293 cells.

The specific aims are:

1. To evaluate the sensitivity of lung cancer cell lines with somatic mutations in B-RAF and STK11 / LKB1, to the highly specific MEK inhibitor, PD0325901. Subsequently, drug-treated lysates from these cell lines will be analyzed by immunoblotting for expression of proteins that regulate the formation of the translation pre-initiation complex, including 4EBP1 and S6 ribosomal protein.

2. To create a library of K-RASG12 and B-RAF466, 469, L597 mutants that are stably expressed in HEK 293 cells. These will also be co-expressed with a truncated variant of STK11 / LKB1. Stable expression of mutant proteins in HEK 293 cells will permit different combinations of mutated proteins to be selected for simultaneously. This library of overexpressing cells will be analyzed to determine the effect of the various mutations on sensitivity to the MEK inhibitor, PD0325901.

To isolate CAP-binding proteins from HEK 293 cells that overexpress full-length and mutant B-RAF and K-RAS mutants, and to compare monosome (untranslated) and polysome-bound (translated) RNA species by gene expression profiling. Bioinformatic analysis will interrogate expression data for differentially translated mRNA species that segregate with either B-RAF, or K-RAS mutation. The effect of the MEK-inhibitor, PD0325901, on the expression of actively translated mRNA’s species for B-RAF or K-RAS mutant genotypes, will also be determined. These data will be used to generate a gene expression ‘signature’ for each genotype that predicts response to MEK-directed therapy. The validity of expression ‘signatures’ will be evaluated by immunoblotting in a panel of B-RAF and K-RAS mutant cell lines derived from adenocarcinomas and bronchioalveolar adenocarcinomas, after treatment with PD0325901.