Search

Pancreatic cancer (PC) is one of the most aggressive types of cancer, with a five-year overall survival rate of 11% among all-comers. Current systemic therapeutic options are limited to cytotoxic chemotherapies which have limited clinical efficacy and are often associated with development of drug resistance. Analysis of The Cancer Genome Atlas showed that wild-type isocitrate dehydrogenase (wtIDH1) is overexpressed in pancreatic tumors. In this study, we focus on the potential roles of wtIDH1 in pancreatic cancer chemoresistance. We found that treatment of pancreatic cancer cells with chemotherapy induced expression of wtIDH1, and this serves as a key resistance factor. The enzyme is protective to cancer cells under chemotherapy-induced oxidative stress by producing NADPH and alpha-ketoglutarate to maintain redox balance and mitochondrial function. An FDA-approved mutant IDH1 inhibitor, ivosidenib (AG-120), is actually a potent wtDH1 inhibitor under a nutrient-deprived microenvironment, reflective of the pancreatic cancer microenvironment. Suppression of wtIDH1 impairs redox balance, results in increased ROS levels, and enhances chemotherapy induced apoptosis in pancreatic cancer vis ROS damagein vitro.In vivoexperiments further revealed that inhibiting wtIDH1 enhances chemotherapy anti-tumor effects in patient-derived xenografts and murine models of pancreatic cancer. Pharmacologic wtIDH1 inhibition with ivosidenib represents an attractive option for combination therapies with cytotoxic chemotherapy for patients with pancreatic cancer. Based on these data, we have initiated phase Ib trial combining ivosidenib and multi-agent chemotherapy in patients with pancreatic cancer (NCT05209074).

Additional file 4: Supplementary Figure 4. IDH1 knockout suppresses tumor growth in vivo. A] Relative mRNA levels, normalized to mRNA levels of 18S; Western blot analysis of IDH1 expression after IDH1 knockout by CRISPR/Cas9 (IDH1.KO) compared to control (IDH1.EV) A375 cells. B] Mice were injected with IDH1.EV and IDH1.KO A375 cells (n = 5 per group) and tumor sizes were monitored for 5 weeks. Images of tumors at the end of the experiment are shown. C] Tumor volumes of IDH1.EV and IDH1.KO A375 xenografts. D] Histograms show tumor volumes with IDH1.EV and IDH1.KO A375 xenografts at the end of the experiment. E] Relative glucose levels in adjacent skin and xenograft. F] Relative free magnesium levels in skin, xenografts and serum. Each data point represents the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Additional file 5: Supplementary Figure 5. In vitro response of melanoma cells to treatment with TMZ in combination with AG-120. A] Silencing IDH1 followed by treatment with TMZ for 5 days under high glucose (25 mM) and B] low glucose (2.5 mM) in SK-MEL-28 cells; IC50 values are provided. C] Relative ROS levels after 48 hours in A375 cell line under low glucose combined with TMZ and AG-120. D] Relative ROS levels after 48 hours in SK-MEL-28 cells under low glucose combined with TMZ and AG-120. E] Cell viability of SK-MEL-28 cells, treated with the indicated doses of TMZ; IC50 values are provided. F] Cell viability of SK-MEL-28 cells treated with indicated doses of AG-120 for 6 days. IC50 values are provided. G] Drug sensitivity in SK-MEL-28 cells under low glucose, with varying concentrations of TMZ and AG-120, cultured for 5 days under low glucose. IC50 results are provided. H] Drug matrix heatmap 5 × 8 (AG-120 and TMZ) grid showing percent viability and Bliss Independence scores in SK-MEL-28 cells cultured under 2.5 mM glucose for 5 days. Positive values reflecting synergism appear green on the heatmap (Bliss volume ≥ 10). All treatments with AG-120 were carried under low glucose (2.5 mM) and low Mg2+ (0.08 mM).

Additional file 3: Supplementary Figure 3. AG-120 is a potential wtIDH1 inhibitor under glucose limitation in melanoma cells. A] Representative images of colony formation assays for cells cultured under high (25 mM) and B] low glucose (2.5 mM) in the SK-MEL-28 cell line. The cells were treated with vehicle control or AG-120 (2 μM) under the indicated conditions for 9 days. Quantitation (%) is shown in the graph at the bottom. C] Cells cultured under high glucose, or D] low glucose were captured by phase-contrast imaging (4X magnification), after treatment with vehicle control or AG-120 (2 μM) for 4 days. Representative oxygen consumption rate (OCR) in A375 E] and SK-MEL-28 F] cell lines cultured in 2.5 mM glucose and treated with vehicle or AG-120 for 36 hours. G] and H] Basal mitochondrial respiration, ATP production, and maximal mitochondrial respiration of the A375 and SK-MEL-28 cells treated with vehicle or AG-120 for 36 hours. Each data point represents the mean ± SEM of three independent experiments. N.S., nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Additional file 6: Supplementary Figure 6. Treatment of mice bearing B16-F10 tumors with TMZ in combination with AG-120. A] Sanger sequencing of PCR amplicons correlated with codon 132 of the IDH1 gene in B16-F10 murine melanoma cells. B] Schematic represents the treatment model after 4 × 104 B16-F10 melanoma murine cells were injected subcutaneously into the flanks of C57BL/6 J recipient mice. After 9 days, when tumors reached 80-100 mm3, mice were divided into four groups and treated with i) Vehicle; ii) TMZ (30 mg/kg intraperitoneal once a day); iii) AG-120 (150 mg/kg orally twice a day); and iv) AG-120 + TMZ (150 mg/kg orally twice a day + 30 mg/kg intraperitoneal daily). C] Survival data of C57BL/6 J mice are represented by Kaplan-Meier curves. Significance between each group was determined using the log-rank test.

Additional file 2: Supplementary Figure 2. IDH1 supports mitochondrial function under glucose limitation. A] Representative OCR tracing in A375 melanoma cells cultured under the indicated glucose concentrations for 24 hours and B] Extracellular acidification rate (ECAR) response of A375 cells under the indicated conditions. C] PCA of metabolites analyzed by LC-MS/MS performed on A375 cells under high (25 mM) and low (2.5 mM) glucose (n = 3 samples). D] A heatmap of the top 50 metabolites with the greatest changes in A375 cells (n = 3 independent samples) under 25 mM glucose. The scale is log 2 fold-change. E] Representative OCR tracing in A375 melanoma cells cultured under the indicated glucose concentrations for 24 hours.