Metabolic Reprogramming By PRDM16 Drives Cytarabine Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) patients with high PRDM16 expression frequently experience induction failure and have a poor prognosis. However, the underlying molecular mechanisms are poorly understood. Here, we show that AML cells with high PRDM16 expression acquire resistance to cytarabine (AraC) through the activation of oxidative phosphorylation (OxPHOS) via regulation of c-MYC.

Initially, we established a murine model of AML with high Prdm16 expression. We transduced c-Kit ⁺ bone marrow cells from wild-type mice with a retrovirus vector encoding MLL-AF9 (MF9) and the oncogenic short form of Prdm16 (sPrdm16). Using MF9/sPrdm16 cells, we next investigated whether AML cells with high Prdm16 expression exhibited resistance to AraC and/or daunorubicin (DNR), chemotherapeutic agents commonly used for induction chemotherapy. In the cell viability assay, MF9/sPrdm16 cells showed significantly higher IC ₅₀ against AraC, but not against DNR, compared to MF9/control (control) cells, suggesting that high sPrdm16 expression confers relative resistance to AraC on AML cells. Colony-forming assay also confirmed resistant to AraC in MF9/sPrdm16 cells compared to control. Moreover, MF9/sPrdm16 cells were also resistant to AraC in vivo as demonstrated by the shortened survival of mice receiving MF9/sPrdm16 cells.

To elucidate the mechanism of AraC resistance, we first examined cell cycle status of MF9/sPrdm16 cells since AraC exerts cytotoxicity in cell cycle dependent manner. To our surprise, MF9/sPrdm16 cells were cycling more rapidly than control cells as demonstrated by decreased G ₀ phase and increased S/G ₂/M phase. Intriguingly, RNA-sequencing analysis revealed that MF9/sPrdm16 cells displayed a signature of high OxPHOS by gene set enrichment analysis (GSEA). Indeed, oxygen consumption rate, superoxide production from mitochondria, and mitochondrial membrane potential were significantly elevated in MF9/sPrdm16 cells compared to control cells. The inhibition of mitochondrial respiration with metformin, the electron transport chain complex I inhibitor, or tigecycline, the mitochondrial protein synthesis inhibitor, abrogated AraC resistance in MF9/sPrdm16 cells by inducing an energetic shift towards a lower OxPHOS status. These results clearly indicated that sPrdm16 overexpression conferred MF9 cells with AraC resistance byincreasing OxPHOS in mitochondria.

Next, we explored the mechanism how sPrdm16 regulates OxPHOS. In addition to elevated OxPHOS signature, RNA-sequencing data also showed strong enrichment of Myc target-signature in MF9/sPrdm16 cells. Western blot analysis showed that sPrdm16 overexpression induced up-regulation of c-Myc and its transcriptional target, Slc1a5, a glutamine transporter. We went on to analyze metabolic changes induced by sPrdm16 overexpression by capillary electrophoresis and mass spectrometry-based metabolome analysis. We found that sPrdm16 induced the accumulation of glutamine, glutamate, α-ketoglutaric acid, succinate, fumarate, and malate in MF9 cells, possibly by activating glutaminolysis via the regulation of c-Myc and Slc1a5 expression, leading to the activation of tricarboxylic acid (TCA) cycle and OxPHOS. We also confirmed the contribution of c-Myc to AraC resistance by overexpressing c-Myc in MF9 cells. As expected, MF9/c-Myc cells mimicked the phenotype of MF9/sPrdm16 cells such as resistance to AraC and high OxPHOS status. Furthermore, 10058-F4, a c-Myc inhibitor, abrogated AraC resistance in MF9/sPrdm16 cells.

Next, we asked if the observation in MF9/sPrdm16 cells could be translated into human AML cells. Human AML cell lines, MOLM-13 and THP-1, with sPRDM16 overexpression also showed significantly higher IC ₅₀ against AraC compared to Mock expressed cells. Moreover, RNA-seq data from the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 clinical trial showed that AML blasts with high PRDM16 expression demonstrated a gene expression signature associated with “OxPHOS” and “Myc Targets”.

In conclusion, our findings demonstrate that PRDM16 overexpression activates mitochondrial respiration through metabolic reprogramming via c-MYC, thereby conferres 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.