Metabolic reprogramming by PRDM16 drives cytarabine resistance in acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML) patients with high PRDM16 expression frequently experience induction failure and have a poor prognosis. However, the molecular mechanisms underlying these clinical features remain elusive. We found that murine AML cells transformed by MLL::AF9 fusion and oncogenic short-isoform Prdm16 overexpression (hereafter, MF9/sPrdm16) exhibited resistance to cytarabine (AraC), but not to anthracycline, both in vitro and in vivo. Intriguingly, MF9/sPrdm16 cells displayed a gene expression signature of high oxidative phosphorylation (OxPHOS) and increased mitochondrial respiration. The inhibition of mitochondrial respiration with metformin or tigecycline abrogated AraC resistance in MF9/sPrdm16 cells via an energetic shift toward low OxPHOS status. Furthermore, sPrdm16 up-regulated Myc and the glutamine transporter Slc1a5, activating the TCA cycle and glutaminolysis. Of note, both OxPHOS and MYC-target gene signatures were significantly enriched in AML patient samples with high PRDM16 expression. Together, we showed that PRDM16 overexpression activates mitochondrial respiration through metabolic reprogramming via the MYC-SLC1A5-Glutaminolysis axis, thereby conferring AraC resistance on AML cells. These results suggest that targeting mitochondrial respiration might be a novel treatment strategy to overcome chemoresistance in AML patients with high PRDM16 expression.

Figure 1.

sPRDM16 induces cytarabine resistance in acute myeloid leukemia cells in a cell cycle-independent manner. (A) Experimental schema for the establishment of MA9/control or MA9/sPrdm16 cells. (B) Protein expression levels of sPrdm16, detected by western blotting analysis, were up-regulated in MA9/sPrdm16 cells. (C) In vitro drug sensitivity assay for cytarabine (upper panel) or daunorubicin (lower panel) using MA9/control and MA9/sPrdm16 (N=3 for each group). (D, E) Drug sensitivity assay using human acute myeloid leukemia (AML) cell lines, MOLM-13 (D) and THP-1 (E), transduced with either mock or sPRDM16-expressing vector (N=3 for each group). (F) Cell cycle analysis of MA9/control and MA9/sPrdm16 cells (N=3 for each group). Representative flow cytometry images (left panels) and percentages of each phase of the cell cycle (right panel) are shown. MA9/sPrdm16 cells exhibited an increased S/G2/M phase, which typically exhibits high sensitivity to cytarabine. All data are represented as mean ± standard deviation. An unpaired Student t test was used to calculate P values. N.S.: not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; *****P<0.00001.

Figure 2.

sPrdm16 activates oxidative phosphorylation. (A) Differentially expressed genes in MA9/sPrdm16 cells compared to MA9/ control cells. There were 7,658 up-regulated genes and 281 down-regulated genes. (B, C) Top 15 gene signatures significantly changed in gene set enrichment analysis (GSEA) using RNA-seq data from MA9/sPrdm16 versus MA9/control cells (B). The gene signature related to “OXIDATIVE PHOSPHORYLATION” was the most significantly enriched in MA9/sPrdm16 cells (B, C). (D) Mito Stress Test (left panel) and Glycolysis Stress Test (right panel) in MA9/control and MA9/sPrdm16 cells (N=3 for each group). (E) Mitochondrial reactive oxygen species (ROS) and relative membrane potential in MA9/ sPrdm16 cells were measured using MitoSOX and the ratio of tetramethylrhodamine methyl ester (TMRM) and mitotracker, respectively (N=3 for each group). All data are represented as mean ± standard deviation. An unpaired Student t test was used to calculate P values. **P<0.01; ***P<0.001; ***P<0.0001. MFI: mean fluorescence intensity.

Figure 3.

Pharmacological inhibition of mitochondrial respiration abrogated cytarabine resistance in MA9/sPrdm16 cells. (A, B) In vitro drug sensitivity assay for cytarabine with vehicle (left panel) or metformin (right panel), an electron transport chain complex I inhibitor (A), and with vehicle (left panel) or tigecycline (right panel), a mitochondrial protein synthesis inhibitor (B). Metformin and tigecycline abrogated cytarabine resistance in MA9/sPrdm16 cells (N=3 for each group). (C) In vitro drug sensitivity assay for cytarabine with vehicle (left panel) or IACS-010759 (right panel), a selective mitochondrial complex I inhibitor. IACS-010759 also abrogated cytarabine resistance in MA9/sPrdm16 cells (N=3 for each group). All data are represented as mean ± standard deviation. An unpaired Student t test was used to calculate P values. N.S.: not significant; ^*P<0.05; ^**P<0.01; ^***P<0.001; ^****P<0.0001.

Figure 4.

sPrdm16 induces metabolic reprogramming through the activation of glutaminolysis via the Myc-Slc1a5 axis. (A) Metabolite concentrations in the TCA cycle of MA9/control or MA9/sPrdm16 cells were measured using capillary electrophoresis time-of-flight mass spectrometry-based metabolome profiling (N=6 for each group). (B) Intracellular glutamine (left panel) and glutamate uptake (right panel) levels, measured by Glutamine/Glutamate-Glo Assay, were higher in MA9/sPrdm16 cells compared to control (N=3 for each group). (C) Transcripts per million (TPM) values of Slc1a5 gene were up-regulated in MA9/sPrdm16 cells (N=3 for each group). (D) Protein expression levels of Slc1a5, detected by western blotting analysis, were also up-regulated in MA9/sPrdm16 cells. (E) The gene signatures related to “MYC-TARGET” were significantly enriched in MA9/sPrdm16 cells. (F) TPM values of Myc gene were up-regulated in MA9/sPrdm16 cells (N=3 for each group). (G) Protein expression levels of Myc, detected by western blotting analysis, were also up-regulated in MA9/sPrdm16 cells. All data are represented as mean ± standard deviation. An unpaired Student t test was used to calculate P values. NS: not significant; ^*P<0.05; ^**P<0.01; ^***P<0.001; ^*****P<0.00001.

Figure 5.

Myc is a key regulator of sPrdm16-mediated resistance to cytarabine. (A) Relative Myc expression in MA9/sPrdm16 cells with Myc knockdown (N=3 for each group). (B) In vitro drug sensitivity assay for cytarabine in MA9/sPrdm16 cells with Myc knockdown (N=3 for each group). (C) In vitro drug sensitivity assay for cytarabine with vehicle or 10058-F4, a Myc inhibitor, in MA9/ control or MA9/sPrdm16 cells (N=3 for each group). In contrast to MA9/control cells, the combination of cytarabine and 10058-F4 reduced the IC₅₀ value of cytarabine in MA9/sPrdm16 cells. (D) Relative Myc expression in MLL::AF9 leukemic cells transduced with Myc-expressing vector (MA9/Myc) (N=3 for each group). (E) Protein expression levels of Myc, detected by western blotting analysis, were also up-regulated in MA9/Myc cells. (F) In vitro drug sensitivity assay for cytarabine in MA9/Myc cells (N=3 for each group). MA9/Myc cells mimicked the phenotype of MA9/sPrdm16 cells as evidenced by the resistance to cytarabine. All data are represented as mean ± standard deviation. An unpaired Student t test was used to calculate P values. NS: not significant; ^*P<0.05; ^**P<0.01; ^***P<0.001; ^****P<0.0001.

Figure 6.

MA9/sPrdm16 mice are resistant to cytarabine. Kaplan-Meier survival curve of MA9/control (left panel) and MA9/sPrdm16 (right panel) mice treated with either vehicle (N=10-11) or cytarabine (N=11). 1.0x10⁴ leukemia cells were transplanted to each sublethally irradiated (6.0 Gy) recipient mouse. MA9/sPrdm16 mice exhibited resistance to cytarabine. The log rank test was used in survival analysis. IP: intraperitoneal injection.

Figure 7.

Primary acute myeloid leukemia patient samples with high PRDM16 expression exhibit gene expression signatures associated with “OXPHOS” and “MYC-Targets”. (A) Relative total PRDM16 expression (including both full-length and short isoform) in acute myeloid leukemia (AML) patients with low (left, N=100) or high (right, N=39) PRDM16 expression. (B) Gene signatures significantly changed in gene set enrichment assay (GSEA) using RNA-seq data from 139 pediatric AML patient samples registered in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 clinical trial. (C) The gene signatures related to “OXIDATIVE PHOSPHORYLATION” (left panel) and “MYC-TARGET” (middle and right panels) were significantly enriched in AML samples with high PRDM16 expression. (D) Proposed model of cytarabine resistance mechanisms in AML with high Prdm16 expression. PRDM16 drives cytarabine resistance through upregulation of mitochondrial respiration via upregulation of MYC and glutamine transporter SLC1A5, activation of glutamine uptake, and glutaminolysis. Therefore, targeting mitochondrial respiration might be a novel treatment strategy to overcome cytarabine resistance in AML patients with high PRDM16 expression. Statistical analyses were performed using the Mann-Whitney U test. ^****P<0.0001.