Identification of somatic mutations in diverse tumor types has grown exponentially with the development of next-generation sequencing technologies. However, there is a pressing need to validate putative driver genes and separate them from coincidental passenger mutations. Further, it is becoming clear that many cancers are highly heterogeneous in terms of the polyclonality of somatic genotypes-often expressing multiple driver mutations simultaneously. Moreover, standard of care treatment often induces selective pressures resulting in significant alterations in recurrent populations. Because of the complex and time-consuming nature intrinsic to generating and utilizing genetically engineered mouse models, we are only in the beginning stages of validating driver genes in many tumor types and are even further behind in studying mechanisms of recurrence in these systems. The central theme of this grant application is to generate a next-generation STRATUM (Somatic Transgenic Tumor Model) toolset for the rapid validation of cancer driver genes and validate them in vivo by expression alone and in combination as demonstrated by patient sequencing. As an initial step, we have pioneered an electroporation-based transposon method for rapid, non-invasive, somatic transgenesis for high throughput validation of tumor driver genes. We will create these novel in vivo models of pediatric glioma as a test case for the utilization of this system for later use with diverse tumor types. Finally, we have incorporated state-of-the-art transgenes for identification, lineage tracing, and ablation of quiescent and recurrent tumor populations. The overall objective of the proposal is to perform advanced development of STRATUM to allow for generalized use in diverse tumor contexts and, therefore, demonstrate the potential of this technology to transform cancer research. We propose to carry out this work in three parts. The focus of Specific Aim 1 is to optimize our STRATUM system for rapid tumor generation to allow for more facile cloning of candidate mutations. The main goal of Specific Aim 2 is to rigorously validate STRATUM in vivo. To accomplish this, each element will be expressed in combination with a validated set of glioma driver genes. Finally, to prepare for widespread dissemination of these tools, in Specific Aim 3 we will generate and validate a ROSA26-targeted construct the generation of Cre recombinase-regulated conditional mouse models. Successful completion of these experiments will create the foundation for a long-lived, cornerstone toolset for understanding both basic, pathologic mechanisms of the disease as well as providing definitive, genetic, proof-of-principle validation of a new therapeutic approach and mechanisms of progression and recurrence in a diverse array of cancers.
Despite the fact that list of cancer-associated mutations has reached almost 1% of the total genome, most mutations do not generate tumors in isolation but require combinatorial expression with other somatic mutations. We propose to validate a modular, state-of-the-art genetic system for the rapid somatic mutation of several candidate genes simultaneously as well as provide a set of extensions for investigating tumor progression and recurrence. If successful, this technology will provide a uniform, powerful, extensible genetic framework from which to interrogate cancer pathomechanisms.
