The discovery that cancer cells harbor mutated proteins necessary for their survival has led to the development of targeted therapies to specifically inhibit them. These highly specific drugs have shown encouraging clinical results, and almost total tumor remission is possible in some instances. However, an inevitable and poorly- understood limitation is the ability of cancer cells to develop resistance, bypassing or blocking the therapies effect. In this proposal, we seek to mechanistically understand how tumors are able to quickly evolve and survive therapy. By mechanistically probing the effects of tumor microenvironment on tumor cells, specifically the phenomenon of hypoxic stress, two novel pathways that increase mutagenesis during low-oxygen stress were discovered. Both of these pathways act on DNA-repair mechanisms, decreasing high-fidelity homologous recombination (HR) repair, and promoting low-fidelity, error-prone non-homologous end joining (NHEJ). This switch to NHEJ is associated with the development of new mutations and copy number variations, both traits of genomic instability and potential mechanisms of therapy resistance. The mechanistic goal of this research proposal is to restore HR repair, thus decreasing the impact of mutagenic NHEJ on tumor progression. In the context of targeted therapy, this could reduce the development of resistance-causing mutations. This will be done by investigating two potential mechanisms by which hypoxia stimulates the HR/NHEJ mutagenic switch.
(Aim 1) We have found that the master transcriptional regulator for hypoxia, HIF-1, mediates the proteasomal degradation of Rad51, a key HR protein, while promoting the activation and expression of NHEJ proteins. The mechanism of the Rad51 degradation will be elucidated, and rescue experiments will be performed using assays for HR function and genomic instability.
(Aim 2) Our bioinformatics analysis of multiple genome-wide RNA sequencing screens has identified 3 novel HIF-1 target genes that are putative negative regulators of HR. Intriguingly, these are all mitochondrial metabolic enzymes, namely spermine synthase, 6-phosphofructo-2- kinase/fructose-2,6-biphosphatase 4, and mannose phosphate isomerase. This novel class of metabolic enzymes, dubbed SIMME, or stress-induced metabolic mutator enzymes, provides a novel connection between stress response in the mitochondria and increased mutagenesis in the nucleus, both promoting cell survival and adaptation following stress; the mitochondria by modulating energy usage, and the nucleus by promoting the accumulation of new mutations. The mechanism by which these enzymes decrease HR repair will be probed using siRNA silencing, protein overexpression, and CRISPR-mediated genetic editing to create knock-outs and point mutants. Finally, the effect of rescuing HR on the sensitivity to targeted therapy will be explored by implanting SIMME mutant tumors in mice, while treating with targeted therapy. The long-term goal of this project is to use these targets and mechanisms to prevent microenvironment-induced mutagenesis, developing agents to prevent or delay the onset of resistance in patients receiving targeted therapy.

Public Health Relevance

Although targeted drugs against cancer have shown tremendous promise in the clinic, a major problem is their inevitable development of resistance. Our studies will explore how cancer cells develop mutations that drive this adaptation, with the ultimate goal of prolonging the therapeutic sensitivity of patients' tumors.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31CA203286-02
Application #
9249392
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Radaev, Sergey
Project Start
2016-04-01
Project End
2021-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
2
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Sanford Burnham Prebys Medical Discovery Institute
Department
Type
DUNS #
020520466
City
La Jolla
State
CA
Country
United States
Zip Code
92037