Developing a Therapeutic Strategy for Targeting Metabolic Vulnerability of TET2 Mutant Leukemia

Background

Loss-of-function TET2 mutations (TET2^MT) occur in up to 30% of Myelodysplastic Syndrome and Acute Myeloid Leukemia. In Chronic Myelomonocytic Leukemia it is > 66%. Additionally, TET2^MT is a founding lesion in myeloid neoplasms (MN), frequently appearing in clonal hematopoiesis of indeterminate potential (CHIP), a prodromal condition that increases the risk for MN and other diseases. Therefore, targeting the founder TET2^MT clone represents a promising strategy to disrupt clonal proliferation at its source. We discovered that the loss of TET2 leads to metabolic rewiring, making cancer cells vulnerable to therapeutic interventions targeting glutamine metabolism (Gu et. al. Blood 2023). CB839, a potent glutaminase inhibitor, effectively disrupts glutaminolysis. However, previous clinical attempts to target glutamine metabolism pathways using CB839 have been unsuccessful. We propose that TET2^MT creates unique metabolic signatures that can be specifically targeted with CB839. Consequently, TET2^MT can serve as biomarkers for glutaminolysis inhibitors in the selective treatment of AML.

Methods and Results

Utilizing CRISPR-Cas9 edited clonal isogenic TET2^WT and TET2^KO cells derived from THP1 (Guan et al., 2021), coupled with HR LC-MS, we identified ~5000 features through untargeted analysis, with significant changes in 810. Targeted analysis confirmed unique metabolic disturbances in amino acid, glutamine, methionine, nicotinamide, and glycolytic pathways in TET2^KO compared to TET2^WT. Pathway enrichment analysis highlighted glutamine/glutamate biosynthesis as the most disturbed pathway. Significantly lower glutamine levels were observed in TET2^WT patient cells and TET2^KO mice compared to wildtype controls. These changes are primarily due to intracellular effects, as reflected in the significantly upregulated expression and activity of glutaminase (GLS1) and glutamate dehydrogenase (GULD1) in TET2^KO cells.

We conducted ¹³C glucose and ¹³C/¹⁵N glutamine metabolic flux analysis with complete carbon and nitrogen atom tracing of Glucose and Glutamine. Media glucose consumption was similar in TET2^KO and TET2^WT cells. However, TET2^KO showed a significant buildup of cellular glucose and a slower consumption rate of glucose-6-phosphate, indicating slower glycolysis. In TET2^WT cells, > 60% of αKG comes from glycolysis, while in TET2^KO cells, it is < 40%, consistent with higher flux from Glutamine due to increased GLS1 and GULD1 activity (Gu et al., Blood 2023). Our data suggest that TET2^MT creates metabolic reprogramming, leading to heightened glutamine addiction that can be therapeutically exploited.

Consistent with our hypothesis, loss of TET2 resulted in high sensitivity to glutamine restriction and treatment with the GLS1 inhibitor CB839 (Jiang, B., et al. Mol Cell 2022). We performed metabolic analysis to understand how CB839 limits cell growth in TET2^KO cells. The most impacted pathway was glutamine metabolism in the mitochondria, where glutamine replenishes the TCA cycle. Glutamic acid levels were reduced more than two-fold with CB839 treatment in TET2^KO compared to TET2^WT. As a result, TET2^KO cells showed a 3-5-fold reduction in αKG with CB839 treatment compared to TET2^WT. Significant reductions in αKG downstream TCA cycle compounds, such as succinate, fumarate, and malate, were observed, with less impact on upstream compounds citrate and oxaloacetate. Inhibition of glutaminase reduced overall downstream metabolites, while IMP, the first product of de novo purine synthesis, increased 2-3-fold. These findings confirm that TET2 loss promotes glutamine metabolic pathway addiction, with CB839 inhibiting TET2^KO cell growth by disrupting the glutamine-dependent TCA cycle.

Given that the TET2 inhibitors Eltrombopag (Epag) and TETi76 mimic the loss of TET2 and create similar disruptions in glutaminolysis, a combination therapy approach for non-TET2 mutated AML using TETi and CB839 can be utilized.

Conclusion.

Our study provides a detailed mechanistic understanding of how TET2 mutations contribute to MN and CHIP. This discovery paves the way for a biomarker-driven, novel therapeutic strategy for myeloid leukemia. Furthermore, our research highlights the potential of combining TET2 inhibitors with glutaminolysis inhibitors as a pioneering treatment approach for a broad range of leukemia types.