KRAS is the single most frequently mutated oncogene in human cancer, yet there are no targeted therapies available for patients whose cancers harbor these mutations. Mutant KRas is intrinsically difficult to target pharmacologically due to a number of factors and is considered by many to be an undruggable protein. An alternative approach to direct targeting is to target proteins to which KRAS mutant tumors have developed a artificial dependency, a condition termed synthetic lethality. Approaches to identifying synthetic lethal relationships in the context of mutant KRas have not been entirely successful; perhaps in part due to their reliance on 2D cell culture, insufficient sensitivity, anda lack of appropriate validation performed in multiple wild-type and mutant KRas systems. This project will take seek to address these weaknesses using KRas mutant lung adenocarcinoma PDXs harboring the most common mutant alleles (G12C (3), G12D (1), G12V (1), and G13D (1)) as well as wild-type KRAS.
The first Aim i s based on development of doxycycline inducible Cas9 nuclease PDX lines and in vivo CRISPR/Cas9 screening. Lung adenocarcinoma PDXs with varying KRAS mutation status will be transduced with an inducible Cas9 lentivirus and selected for expression of a constitutive reporter gene to obtain a pure population of transduced cells. These lines will be transduced with a pool of lentiviral sgRNAs targeting 19,050 human genes and xenografted into nave mice. After tumors are established, a portion of each tumor will be excised, followed by induction of Cas9 expression by ad libitum doxycycline medicated chow. sgRNA frequencies will be compared across multiple tumors at baseline and after several weeks of in vivo selection.
The second Aim uses an orthogonal approach to target validation in lung adenocarcinoma PDXs based on doxycycline inducible shRNAs and serial measurement of secreted luciferase in blood as a marker of clonal dynamics in vivo. In an orthogonal approach, lung adenocarcinoma PDXs will be independently transduced with lentiviral constructs that inducible express one of two secreted luciferases and either a control or gene targeting shRNA. After tumors are establish, luciferase and shRNA co-expression will be induced by doxycycline and subclonal tumor population dynamics measured by frequent collection of small volumes of peripheral blood. Deliverables from these studies are expected to include 1) doxycycline inducible Cas9 lentiviral vectors suitable for PDX transduction and positive selection; 2) lung adenocarcinoma PDX lines representing the most common KRas mutant alleles bearing these constructs; 3) candidates for synthetic lethal interactions in the context of KRAS mutations; 4) A novel platform for cross-validating our own synthetic lethal candidates and those of the KRAS Synthetic Lethality Network in lung adenocarcinoma PDXs harboring the most common KRAS mutant alleles.
KRAS mutations are the single most common driver oncogenes in human cancer. KRAS is intrinsically difficult to target with new drugs. The KRAS Synthetic Lethality Network intends to identify synthetic gene dependencies in KRAS mutant cancers. We are taking 2 approaches to help this effort: first using patient derived xenografts and in vivo loss of function screening, second using secreted reporter enzymes to track clonal tumor dynamics and to cross-validate the findings of other researchers in the Network. These approaches will identify strategies to treat KRAS mutant cancers.
