Introduction

Tumor progression and survival is driven by genetic mutations and environmental conditions. Recent work has revealed that redox metabolism is deregulated in pancreatic cancer and may provide new opportunities for diagnosis and targeted treatment. As such, recent studies have sought to understand how changes in metabolic flux in response to oxidative stress can be leveraged for the targeted treatment of cancer. Specifically, a therapeutic strategy for targeting KRAS mutant cancers using high dose ascorbate was introduced to disrupt redox homeostasis.¹ Ascorbate can be oxidized in the presence of Reactive Oxygen Species (ROS) to form dehydroascorbate (DHA), which is transported via glucose transporter (GLUT1). After entering the cell, DHA is reduced back to ascorbate, which causes a depletion of the intracellular redox machinery and in turn sensitizes cells to ROS. Each successive turn of this process results in a cyclical increase in oxidative stress in the tumor microenvironment leading to downstream inhibition of DNA damage repair enzymes. Within this therapeutic approach, it is known that enzymes in the DNA damage response pathway can potentially modulate flux by upregulating the pentose phosphate pathway (PPP). While high dose ascorbate is of great interest as an oxidative stress-mediated therapeutic approach for cancer, assessing this therapeutic strategy and its downstream metabolic targets has been challenging. Here, we report hyperpolarized DHA (HP DHA) as a metabolic biomarker for monitoring oxidative stress in vivo, leading to the discovery that high dose ascorbate therapy increases flux through the PPP in cancer.

Methods

Ascorbate therapy of pancreatic cancer PDXs – BRAF-driven (PC87), BRCA-driven (HyMad1) and KRAS-driven (PC106) PDX models, created from patient samples and verified using targeted IMPACT sequencing,^4,5 were subcutaneously xenografted in the right flank of NSG mice. 2 weeks after implantation, mice were randomly divided into two groups. One group was treated with freshly prepared vitamin C in 400 μL of PBS (4 g/kg) twice a day via IP injection (PC106, n = 7; HyMad1, n = 7). Control group mice were treated with PBS using the same twice a day dose (PC106, n = 7; HyMad1 n = 7). T₂ images of these mice were acquired weekly to quantify tumor size and evaluate changes in tumor growth over time. HP DHA imaging of pancreatic cancer PDXs – Ascorbate and vehicle-treated mice of both tumor models were injected with 250 μL of 30 mM HP DHA in D₂O to investigate the redox status of the implanted tumor over time. ¹³C HP MRS was performed using an axial 2D CSI acquisition on a 5T small animal MRI spectrometer to measure DHA/ascorbate metabolism. DHA/ascorbate ratios were quantified by taking the area under the curve and compared between ascorbate-treated and vehicle mice to determine changes in tumor redox upon ascorbate therapy. Metabolic profiling of glucose consumption – Mouse embryonic fibroblasts (MEFs) with ATM and CHEK2 mutations were treated for 4 hours in RPMI media supplemented with 25 mM [1,2-¹³C₂]-glucose, 100 μM sodium ascorbate and 10% dialyzed FBS (N=3). Cells treated with vehicle were supplemented with an equal volume of phosphate buffered saline instead of 100 uM sodium ascorbate (N=3). 500 μL of media was taken from each well and mixed with 100 μL D₂O with 1 mM ¹³C urea used as reference for chemical shift and concentration determination. ¹³C experiments were acquired to determine the relative peak area under the curves (AUC) for C₃-labeled lactate, C₂,C₃-doubly labelled lactate, and ¹³C urea. Absolute PPP activity and glycolytic activity was then calculated based on measured cell count for each well.

Results and Discussion

High dose ascorbate therapy drives oxidate stress, leading to positive treatment response in a BRCA-mutant and KRAS-mutant model of cancer. Ascorbate treatment was shown to slow tumor growth in a KRAS-driven PDX model after 3 weeks while the effect on tumor growth was significant in a BRCA-driven PDX model after 4 days (Figure 1A). The observed ratios of HP ascorbate/total carbon match with the observed treatment response after 1 week in all 3 models (Figure 1B). Metabolomic studies using mass spectroscopy showed decreased levels of PPP intermediates in the rapidly responding BRCA PDX model, revealing that ascorbate therapy-mediated oxidative stress drives increased flux through the PPP (Figure 2). Metabolic profiling of glucose consumption shows increased PPP activity upon ascorbate treatment in wildtype and DNA damage repair mutant MEFs (Figure 3). Interestingly, ATM-mutant and CHK-mutant MEFs showed lower levels of glycolytic activity and baseline PPP activity than the parent wildtype MEFs. These results, in concert with the observed readouts of oxidative stress, demonstrate HP DHA as an effective biomarker for oxidative stress in tumors.

Conclusion

We demonstrated HP DHA as an imaging agent for probing the biochemical mechanism of response to ascorbate therapy in PDX models of cancer. Changes in DHA/ascorbate metabolism were observed in these tumor models after prolonged treatment, mirroring the observed treatment response to high dose ascorbate. Metabolomic studies then showed that high dose ascorbate therapy induced oxidative stress, leading to increased flux through the PPP. DNA damage repair mutants exhibit lower baseline flux through the PPP and are more sensitized to ascorbate induced ROS. These results demonstrate a new method for assessing oxidative stress in vivo using HP DHA.

Acknowledgements

The authors acknowledge the Stand Up to Cancer Foundation, Starr Cancer Consortium, Thompson Family Foundation, NIH/NCI Cancer Center Support Grant P30 CA008748 and NIH R01 CA 252037 for funding and support.

References

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