Despite immense effort over the past decade to identify therapeutic opportunities based on metabolic reprogramming in adult cancers, very little effort has focused on pediatric tumors. Pediatric tumors are strong candidates for metabolic therapy, because they tend to be genetically simpler than adult cancers, and because there is already a strong basis for clinically-actionable nutrient addictions in pediatric cancer.

For example, L-Asparaginase, which has been used for decades in acute pediatric leukemia’s, works by starving tumor cells of asparagine. Asparagine is conventionally viewed as a non-essential amino acid, but leukemia cells have such a great demand for it that they can be starved and killed by using L-Asparaginase to deplete the plasma supply.

We believe that other forms of childhood cancer display actionable metabolic vulnerabilities, and this User Group seeks to characterize the metabolism of pediatric sarcomas. The research activities sponsored by the CRCFPO services capitalize on a number of strengths at UT Southwestern, including an outstanding team of clinical pediatric oncologists, expertise in cancer metabolism, and a CPRIT-funded multi-investigator grant (MIRA) focused on the genomics and therapeutic liabilities of sarcoma.

Teamed with the CRCFPO’s Component of Chemistry & Radiochemistry, this User Group is characterizing the metabolism of sarcoma cells in culture and in vivo to identify activities that could be used to develop novel PET agents or provide therapeutic leverage. These efforts synergize with the MIRA in that the discovery of novel genetic drivers within the MIRA would immediately trigger examination of the metabolic effects of those drivers, and then the development of novel PET probes, under the interactions between the CRCFPO and the User’s research groups.

Furthermore, metabolic enzymes defined as key vulnerabilities through functional genomics in the MIRA are being studied in much greater detail using the analytical and imaging approaches that has been made available by the CRCFPO.

The approach to understand metabolism in sarcoma capitalizes on several strategies used previously by the teams led by https://profiles.utsouthwestern.edu/profile/99018/ to identify and exploit reprogrammed pathways in other forms of pediatric cancers:

First, comprehensive metabolic analysis is being performed in sarcoma-derived cell lines. These approaches include both targeted and global metabolomic profiling to characterize steady-state metabolite levels. Metabolic flux studies are being performed using ¹³C- and or ¹⁵N-labeled nutrients as described previously. Nutrient deprivation studies (glucose, glutamine) are also being used to determine oncogene-regulated dependence on these fuels. Wherever possible, isogenic cell lines containing and lacking oncogenic drivers are being studied in parallel to define specific effects of each mutation on metabolic reprogramming.

The preliminary observations by the Deberardinis team suggest that the oncogenic fusion EWS-FLI1 in Ewing sarcoma reprograms metabolism to allow cells to resist periods of glucose deprivation; this appears to involve enhanced glycogen deposition in EWS-FLI1-transformed cells. This observation opened up the possibility of using PET radiotracers to observe glycogen deposition in live tumors. One of the team members (DeBerardinis) was involved in the development of such a probe that has already been used in mouse models of cancer.

Second, to analyze metabolism in intact tumors, patient-derived xenografts (PDXs) and cell-line derived xenografts are being used. Wherever possible, conditional silencing of oncogenic drivers is being used to observe the effects of these oncogenes on metabolic reprogramming in vivo. Metabolites extracted from the tumors are being used for metabolomics analysis. Metabolic flux is then being analyzed by infusing mice with [U-¹³C]glucose, [U-¹³C]glutamine and/or other labeled nutrients informed by the metabolomics assessment.

The results of these in vivo studies inform the design of customized PET probes to image reprogrammed metabolic pathways. Plausible examples well within the expertise of the radiochemistry core include ¹¹C or ¹³N labeling of simple nutrients such as acetate, glutamine, or branched-chain amino acids.

Third, we are one of only two groups in the world to have successfully used stable isotope tracers to assess metabolic flux in living human tumors. This cutting-edge technique has enabled us to identify metabolic features of human tumors in situ and – crucially – to identify metabolic differences between cultured cell models and bona fide tumors. This has allowed us to focus ongoing discovery work on pathways with documented relevance to human cancer in vivo. Our approach is to validate altered pathways observed in culture xenograft models or pediatric patients. Validated pathways (e.g., enhanced uptake of amino acids) then prompts clinical studies to examine the utility of PET based on these nutrients, most of which have been established and first tested in mice.

As another example, Dr. Stephen Skapek in this User Group is co-leading a SPORE project that focuses on Malignant Peripheral Nerve Sheath Tumor (MPNST), one of the most common soft tissue sarcomas in children. Although MPNST can represent a sporadic neoplasm, it often occurs in children with neurofibromatosis, in which the sarcoma arises from within a pre-malignant tumor known as a plexiform neurofibroma. Because MPNST is incurable when it cannot be resected or after it has metastasized, early detection of MPNST from within a pre-existing plexiform neurofibroma represents a major challenge.

The aforementioned SPORE project is trying to utilize novel PET radiotracers to both serve as early response markers for the application of targeted therapeutics to MPNST and also to facilitate early detection of sub-clinical MPNST in at-risk individuals. Physicians in our pediatric neurofibromatosis program, one of the largest in the U.S., evaluate over 100 new children each year and actively follow over 450 children known to have neurofibromatosis. With this large clinical program, we are poised to conduct pilot studies in that subset undergoing biopsy or surgical resection of a suspected MPNST to identify PET-based biomarkers. We are therefore focused on using [¹⁸F]FLT-PET, as a biomarker of cell proliferation. However, we will evaluate others, such as [¹¹C]choline or [¹⁸F]choline, and [¹⁸F]ISO-1, as alternatives based on histopathology of these lesions.

Clinically, [¹⁸F]FDG-PET has been used widely to probe glucose uptake and metabolism in tumors. However, its sensitivity is limited to detect lesions less than 0.5 cm and it has high false-positive rate due to inflamed lymph nodes. The CRP provides an opportunity to produce a wider range of radiotracers that can image additional aspects of tumor biology and metabolism. Hyperpolarized ¹³C MRI methods have been shown to probe “pathway specific” aspects of the biochemistry and metabolism of tumors in vivo. To date there have been only a handful of reports combining use of these two approaches in oncology. Drs. Dean Sherry (CRP’s Steering Committee) and Craig Malloy in the Advanced Imaging Research Center have been involved in the development and application of ¹³C hyperpolarization MR studies in both preclinical and human studies.

We have a 3T wide bore human MRI scanner dedicated in part to clinical translational studies in oncology using hyperpolarized ¹³C molecules. The availability of this scanner and the expertise to carry out clinical translational hyperpolarized ¹³C studies has created an opportunity to perform complementary [¹¹C]-PET and [¹³C]-MRI studies in both preclinical models and in humans using a variety of [¹¹C] radiotracers that probe specific aspects of tumor biology and metabolism. A partial list of potential complementary studies using [¹¹C] radiotracers and ¹³C hyperpolarized agents include the pairs of:

• [¹¹C]pyruvate and [¹³C]pyruvate (PDH, LDH, ALT)
• [¹¹C]lactate and [¹³C]lactate (PDH, little metabolism observed in vivo)
• [¹¹C]acetate and [¹³C]acetate (acetyl CoA and acetyl carnitine)
• [¹¹C] or [¹³N]glutamine and [¹³C]glutamine (glutaminase)
• [¹¹C]choline17 and [¹³C or ¹⁵N]choline (lipid synthase, choline kinase)
• [¹¹C]bicarbonate and [¹³C]biocarbonate (extracellular pH, carbonic anhydrase)