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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants Phase II (R33)
Project #
5R33CA202900-03
Application #
9460474
Study Section
Special Emphasis Panel (ZCA1)
Program Officer
Li, Jerry
Project Start
2016-05-01
Project End
2019-04-30
Budget Start
2018-05-01
Budget End
2019-04-30
Support Year
3
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Cedars-Sinai Medical Center
Department
Type
DUNS #
075307785
City
Los Angeles
State
CA
Country
United States
Zip Code
90048
Akhtar, Aslam Abbasi; Gowing, Genevieve; Kobritz, Naomi et al. (2018) Inducible Expression of GDNF in Transplanted iPSC-Derived Neural Progenitor Cells. Stem Cell Reports 10:1696-1704
Zonis, Svetlana; Breunig, Joshua J; Mamelak, Adam et al. (2018) Inflammation-induced Gro1 triggers senescence in neuronal progenitors: effects of estradiol. J Neuroinflammation 15:260
Garrett, Matthew; Sperry, Jantzen; Braas, Daniel et al. (2018) Metabolic characterization of isocitrate dehydrogenase (IDH) mutant and IDH wildtype gliomaspheres uncovers cell type-specific vulnerabilities. Cancer Metab 6:4
Hoang-Minh, Lan B; Dutra-Clarke, Marina; Breunig, Joshua J et al. (2018) Glioma cell proliferation is enhanced in the presence of tumor-derived cilia vesicles. Cilia 7:6
Xu, Liang; Chen, Ye; Dutra-Clarke, Marina et al. (2017) BCL6 promotes glioma and serves as a therapeutic target. Proc Natl Acad Sci U S A 114:3981-3986
Akhtar, Aslam Abbasi; Sances, Samuel; Barrett, Robert et al. (2017) Organoid and Organ-On-A-Chip Systems: New Paradigms for Modeling Neurological and Gastrointestinal Disease. Curr Stem Cell Rep 3:98-111
Akhtar, Aslam Abbasi; Breunig, Joshua J (2017) Tetracycline-Inducible and Reversible Stable Gene Expression in Human iPSC-Derived Neural Progenitors and in the Postnatal Mouse Brain. Curr Protoc Stem Cell Biol 41:5A.9.1-5A.9.12