Determining the Metabolic and Therapeutic Consequences of HGPRT Inhibition in DIPG

Diffuse intrinsic pontine gliomas (DIPG) are aggressive pediatric brain cancers with poor outcomes attributed to treatment resistance. Radiation therapy (RT) extends patient survival, but only on a scale of weeks to months. As a result, patients typically succumb to DIPG within two years of diagnosis. Therefore, it is critical to identify new methods to increase RT efficacy. We reported that DIPG tumors have altered purine metabolism programs characterized by an overreliance on the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) to produce guanylates. We found that abundant guanine within DIPG tumors prevented radiosensitivity induced by the de novo guanine synthesis inhibitor mycophenolic acid. Lastly, we found that HGPRT knockdown was sufficient to radiosensitize DIPG tumors in mice. These findings suggest that HGPRT promotes efficient DNA damage repair following RT in DIPG. The mechanism behind these phenomena is not yet fully understood. I hypothesize that disruption of HGPRT metabolic function impairs RT- induced DNA damage repair in DIPG, leading to increased RT efficacy.

I have developed three aims to test this hypothesis. First, I will test the metabolic consequences of HGPRT inhibition in DIPG cells with stable isotope tracing to track the flow of metabolites as they are used by the cells. This will be accomplished using standard mass spectrometry techniques and with our state-of-the-art Matrix-Assisted Laser Desorption Ionization (MALDI) spatial mass spectrometer which will allow me to visualize DIPG metabolism as it exists within the native tissue architecture. Second, I will determine the effect of HGPRT inhibition on DNA damage repair in irradiated DIPG cells. I will measure DNA damage repair signaling cascades and physical DNA breaks caused by RT in HGPRT-knockdown isogenic models. Lastly, I will measure altered DNA damage repair kinetics by measuring gamma-H2AX, a canonical marker of DNA damage, in DIPG cells and tissues. Third, I will use novel HGPRT inhibitors to measure their ability to increase RT efficacy in DIPG models.

Treatment resistance is the largest barrier to improved outcomes for DIPG patients. RT remains the standard-of-care because it routinely provides some survival benefit when most new strategies provide none. The work outlined here will test a new approach to improve RT efficacy by targeting HGPRT, which has already been shown to play an unknown role in radiosensitization. I will determine the consequences of HGPRT inhibition; mechanistically by measuring the impairment of metabolic and DNA damage response programs and phenotypically by measuring the effect of HGPRT inhibition in improving RT efficacy. Further, testing novel pharmacological inhibitors of HGPRT has the potential to kickstart development of new clinical studies in DIPG patients. Together, the methods discussed in this proposal can identify treatment strategies to combat treatment resistance in DIPG tumors.