Metabolic requirement for GOT2 in pancreatic cancer depends on environmental context.

Mitochondrial glutamate-oxaloacetate transaminase 2 (GOT2) is part of the malate-aspartate shuttle, a mechanism by which cells transfer reducing equivalents from the cytosol to the mitochondria. GOT2 is a key component of mutant KRAS (KRAS*)-mediated rewiring of glutamine metabolism in pancreatic ductal adenocarcinoma (PDA). Here, we demonstrate that the loss of GOT2 disturbs redox homeostasis and halts proliferation of PDA cells in vitro. GOT2 knockdown (KD) in PDA cell lines in vitro induced NADH accumulation, decreased Asp and α-ketoglutarate (αKG) production, stalled glycolysis, disrupted the TCA cycle, and impaired proliferation. Oxidizing NADH through chemical or genetic means resolved the redox imbalance induced by GOT2 KD, permitting sustained proliferation. Despite a strong in vitro inhibitory phenotype, loss of GOT2 had no effect on tumor growth in xenograft PDA or autochthonous mouse models. We show that cancer-associated fibroblasts (CAFs), a major component of the pancreatic tumor microenvironment (TME), release the redox active metabolite pyruvate, and culturing GOT2 KD cells in CAF conditioned media (CM) rescued proliferation in vitro. Furthermore, blocking pyruvate import or pyruvate-to-lactate reduction prevented rescue of GOT2 KD in vitro by exogenous pyruvate or CAF CM. However, these interventions failed to sensitize xenografts to GOT2 KD in vivo, demonstrating the remarkable plasticity and differential metabolism deployed by PDA cells in vitro and in vivo. This emphasizes how the environmental context of distinct pre-clinical models impacts both cell-intrinsic metabolic rewiring and metabolic crosstalk with the TME.

Serial H3K27M cell-free tumor DNA (cf-tDNA) tracking predicts ONC201 treatment response and progression in diffuse midline glioma.

BACKGROUND: Diffuse Midline Glioma (DMG) with the H3K27M mutation is a lethal childhood brain cancer, with patients rarely surviving 2 years from diagnosis. METHODS: We conducted a multi-site Phase 1 trial of the imipridone ONC201 for children with H3K27M-mutant glioma (NCT03416530). Patients enrolled on Arm D of the trial (n = 24) underwent serial lumbar puncture for cell-free tumor DNA (cf-tDNA) analysis and patients on all arms at the University of Michigan underwent serial plasma collection. We performed digital droplet polymerase chain reaction (ddPCR) analysis of cf-tDNA samples and compared variant allele fraction (VAF) to radiographic change (maximal 2D tumor area on MRI). RESULTS: Change in H3.3K27M VAF over time ("VAF delta") correlated with prolonged PFS in both CSF and plasma samples. Nonrecurrent patients that had a decrease in CSF VAF displayed a longer progression free survival (P = .0042). Decrease in plasma VAF displayed a similar trend (P = .085). VAF "spikes" (increase of at least 25%) preceded tumor progression in 8/16 cases (50%) in plasma and 5/11 cases (45.4%) in CSF. In individual cases, early reduction in H3K27M VAF predicted long-term clinical response (>1 year) to ONC201, and did not increase in cases of later-defined pseudo-progression. CONCLUSION: Our work demonstrates the feasibility and potential utility of serial cf-tDNA in both plasma and CSF of DMG patients to supplement radiographic monitoring. Patterns of change in H3K27M VAF over time demonstrate clinical utility in terms of predicting progression and sustained response and possible differentiation of pseudo-progression and pseudo-response.

GOT1 inhibition promotes pancreatic cancer cell death by ferroptosis.

Cancer metabolism is rewired to support cell survival in response to intrinsic and environmental stressors. Identification of strategies to target these adaptions is an area of active research. We previously described a cytosolic aspartate aminotransaminase (GOT1)-driven pathway in pancreatic cancer used to maintain redox balance. Here, we sought to identify metabolic dependencies following GOT1 inhibition to exploit this feature of pancreatic cancer and to provide additional insight into regulation of redox metabolism. Using pharmacological methods, we identify cysteine, glutathione, and lipid antioxidant function as metabolic vulnerabilities following GOT1 withdrawal. We demonstrate that targeting any of these pathways triggers ferroptosis, an oxidative, iron-dependent form of cell death, in GOT1 knockdown cells. Mechanistically, we reveal that GOT1 inhibition represses mitochondrial metabolism and promotes a catabolic state. Consequently, we find that this enhances labile iron availability through autophagy, which potentiates the activity of ferroptotic stimuli. Overall, our study identifies a biochemical connection between GOT1, iron regulation, and ferroptosis.

Tissue of origin dictates GOT1 dependence and confers synthetic lethality to radiotherapy.

BACKGROUND: Metabolic programs in cancer cells are influenced by genotype and the tissue of origin. We have previously shown that central carbon metabolism is rewired in pancreatic ductal adenocarcinoma (PDA) to support proliferation through a glutamate oxaloacetate transaminase 1 (GOT1)-dependent pathway. METHODS: We utilized a doxycycline-inducible shRNA-mediated strategy to knockdown GOT1 in PDA and colorectal cancer (CRC) cell lines and tumor models of similar genotype. These cells were analyzed for the ability to form colonies and tumors to test if tissue type impacted GOT1 dependence. Additionally, the ability of GOT1 to impact the response to chemo- and radiotherapy was assessed. Mechanistically, the associated specimens were examined using a combination of steady-state and stable isotope tracing metabolomics strategies and computational modeling. Statistics were calculated using GraphPad Prism 7. One-way ANOVA was performed for experiments comparing multiple groups with one changing variable. Student's t test (unpaired, two-tailed) was performed when comparing two groups to each other. Metabolomics data comparing three PDA and three CRC cell lines were analyzed by performing Student's t test (unpaired, two-tailed) between all PDA metabolites and CRC metabolites. RESULTS: While PDA exhibits profound growth inhibition upon GOT1 knockdown, we found CRC to be insensitive. In PDA, but not CRC, GOT1 inhibition disrupted glycolysis, nucleotide metabolism, and redox homeostasis. These insights were leveraged in PDA, where we demonstrate that radiotherapy potently enhanced the effect of GOT1 inhibition on tumor growth. CONCLUSIONS: Taken together, these results illustrate the role of tissue type in dictating metabolic dependencies and provide new insights for targeting metabolism to treat PDA.