Ex Vivo Experiments Show That IDH1/2-Mutant Inhibitors Can be Safely Used As Adjuvants to Regular Chemotherapy in IDH1/2-Mutated Acute Myeloid Leukemia

Background: Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2^MT) occur in up to 20% of MDS, MDS/MPN overlap and (s)AML. These mutations induce a metabolic rewiring of cancer cells that may impose metabolic vulnerabilities which can be pharmacologically targeted. In carcinoma cells, IDH1^MT sensitize cells to oxidative stress induced by irradiation and treatment with electron transport chain complex I inhibitors, such as phenformin and metformin. Conversely, IDH1^MT inhibitors protect IDH1^MT cells against these treatment modalities. As a consequence, IDH1^MT inhibitors and cytotoxic drugs should not be used together in patients with IDH1^MT carcinoma and this has clinical relevance since IDH1/2^MT inhibitors are already in phase I clinical trials. The underlying mechanism is that IDH1^MT dramatically reduce the cellular production capacity of NADPH, an important antioxidant, and IDH1^MT inhibitors restore the NADPH production capacity. Of note, IDH1 and IDH2 are the most important providers of NADPH in most cells.

Methods: Primary IDH1/2^MT, IDH1/2^WT and TET2^MT AML cells (n=15) were cultured and treated with the IDH1^MT inhibitor AGI-5198, the IDH2^MT inhibitor AGI-6780, irradiation, phenformin, metformin, daunorubicin or cytarabine. Cell numbers were counted 5 days after therapy exposure. In addition, cells were adhered to microscopy slides and maximal IDH1/2 and G6PD activity was determined using quantitative enzyme cytochemistry. ROS levels were fluorometrically measured using flow cytometry.

Results: We observed that IDH1/2^MT do not sensitize primary AML cells to irradiation, phenformin, metformin, daunorubicin or cytarabine and IDH1/2^MT inhibitors had no effect on therapy sensitivity in primary AML cells. Because it is argued that inactivating TET2^MT function downstream of IDH1/2^MT, TET2^MT AML cells served as a negative control and showed no sensitization neither. Although IDH1/2^MT greatly reduce the IDH1/2-mediated NADPH production capacity in AML cells (P < 0.0001), IDH1/2 are not important NADPH providers in myeloid cells and the contribution of glucose-6-phosphate dehydrogenase (G6PD) to the total NADPH production pool is much larger (maximal G6PD activity is ~4 times higher than maximal IDH1/2 activity, Fig. 1). This contrasts the situation in glioblastoma, where IDH1/2 provide 65% of all NADPH. Large-scale mRNA-sequencing in AML and glio(blasto)ma datasets revealed no differences in IDH1/2 and G6PD expression between types of cancer, suggesting that post-translational modifications perform key roles in regulating the maximal activity of these enzymes (Fig. 2). To investigate the implications of these results on cellular redox potentials, we performed ROS measurements after treatment with irradiation and observed that IDH1/2^MT do not increase ROS in AML cells, whereas IDH1/2^MT do increase ROS in glioma cells.

Discussion: Increased sensitivity to oxidative stress is not a metabolic vulnerability in IDH1/2^MT AML and this is likely due to the fact that maximal NADPH production capacity is affected to only a small extent in IDH1/2^MT AML. Therapies that increase oxidative stress, such as irradiation and metformin, counteract with IDH1/2^MT inhibitors in glio(blasto)ma cells because there, IDH1/2^MT have profound implications on cellular NADPH production capacities and ROS levels. Our results suggest that IDH1/2^MT do not induce sensitivity to cytotoxic drugs in AML cells and that IDH1/2^MT inhibitors can be safely used adjuvantly to the AML standard of care daunorubicin and cytarabine, or whole-body irradiation in the context of bone marrow transplantations. These data are crucial for phase II and III clinical trial designs ofIDH1/2^MT inhibitors.

Fig. 1. IDH1^MT reduce maximal IDH1/2 activity, but IDH1/2 is a more important NADPH producer in glioblastoma than in AML compared with G6PD.

G6PD (a, c, e, g) and IDH1 activity (b, d, f, h) staining of IDH1^R132 non-mutated (a, b, e, f) and mutated (c, d, g, h) primary AML cells (a-d) and glioblastoma (e-h) cryostat sections. The amount of blue color (nitro BT-formazan) directly reflects IDH1/2 and G6PD activity (production of NADPH).

Fig. 2. mRNA sequencing reveals no differences in IDH1, IDH2 and G6PD expression between AML, low-grade glioma (LGG) and glioblastoma (GBM).