Mutant tp53 Causes a Gain of Function Increase in Sensitivity to Reactive Oxygen Species in Erythroid Precursors
Authors: Lund Troy C. , Carter Michelle L , Kramer Ashley C. , Blazar Bruce R. , Ross Julie A. 
Published: November 2019

Abstract
TP53 is mutated in approximated 10 – 15% of cases of leukemia and myelodysplastic syndrome. It is recently appreciated that oxidative stress plays a role in the dysplastic processes that drives these diseases. Animal modeling of oxidative stress has been a challenge as many of the central genes in the oxidative stress response pathway are necessary for life and knockout animals die as embryos due to overwhelming oxidative stress. The zebrafish model allows pro-oxidant exposure early in hematopoietic development, and gata1DsRed1 transgenic animals allow clear identification of erythroid precursors. We capitalized on these advantages to interrogate the effects of oxidative stress on erythroid precursors. The gata1DsRed1 erythroid precursors showed a dose-responsive increase in reactive oxygen species (ROS) generation after naphthol exposure. Pro-oxidant exposure significantly up-regulated several anti-oxidant genes including: hypoxia-inducible factor (hif1a), nuclear factor (erythroid derived)-like 2 (nrf2), ferritin heavy chain (fth1a), thioredoxin (txn), and heme oxygenase 1 (hmox1); (p < 0.05) shown by qRT-PCR. In silico promoter analysis of these genes revealed several tp53 binding sites within 4 kb upstream of the first exon in each gene. Pro-oxidant exposure was able to induce tp53 expression 3-fold over baseline by qRT-PCR. We next took advantage of the tp53 mutant line tp53M214K/M241K which has a mutation in the DNA binding region of tp53 rendering it non-functional. We found that tp53M214K/M124K fish were highly sensitive to pro-oxidant exposure with 80% of embryos showing severe to moderate anemia and cardiac edema after 72 hours of exposure to naphthol (versus 25% in wild-type control animals, n = 100/group; p < 0.001). There was a 3-fold decrease in the number of hemoglobin producing cells in naphthol treated tp53M214K/M124K animals as shown by o-dianisidine staining (versus control animals, n = 10/group; p < 0.01). A dose-response between the amount of pro-oxidant exposure and severity of anemia/edema also existed as determined by correlation of pro-oxidant concentration to an edema severity scale. We next measured the amount of ROS generated after naphthol exposure using CellROX detection assays and found that tp53M214K/M124K animals showed a 5 – 10-fold increase in ROS generated compared to wild-type (n = 30/group, p < 0.01). This increased ROS was maintained even in the heterozygous states of tp53M214K/wt suggesting a gain-of-function phenomenon. ROS generation could be completely reversed by treatment with the anti-oxidant n-acetylcysteine. When we completely abrogated mutant tp53 by morpholino knockdown, ROS generation and hemolysis were significantly decreased, providing further evidence that the ROS generation is a gain-of-function phenomenon of mutated tp53. Finally, uncoupling of mitochondrial respiration using oligomycin also decreased ROS generation by 50% (p < 0.01) implicating a dysregulatory process ongoing in mitochondria. In addition to the recently appreciated increased tumorigenicity caused by mutated tp53, our data suggest that cells harboring mutant tp53 also have increased ROS generation and increased sensitivity with pro-oxidant exposure. Understanding the mechanisms by which the anti-oxidant response is regulated will allow us to potentially find more effective druggable targets to treat the oxidative stress that accompanies the dysplastic process.

Disclosures
No relevant conflicts of interest to declare.