AML Cells Have Increased Mitochondrial Mass but Less Reserve in Their Respiratory Chain Complexes Leading to Heightened Sensitivity to Inhibition of Mitochondrial Protein Translation,

Abstract 3585

Recent studies suggest that dysregulated mitochondrial oxygen consumption promotes the growth of AML cells. Therefore, we characterized the structure and metabolic function of the mitochondria in AML and normal G-CSF-mobilized hematopoietic mononuclear cells (PBSCs). Compared to PBSCs, 1^o AML cells had increased mitochondrial mass as demonstrated by an increased mitochondrial DNA copy number and increased activity of matrix enzyme citrate synthase. The increased mitochondrial mass observed in 1^o AML cells may represent larger mitochondria and/or more numerous mitochondria. Therefore, we evaluated the mitochondria of 1^o AML and normal CD34+ hematopoietic cells by electron microscopy. The mitochondria in 1^o AML cells were larger in area, but fewer in number compared to normal CD34+ cells.

Mitochondria contain the respiratory chain complexes that promote oxidative phosphorylation. Given the dysregulated mitochondrial biogenesis in 1^o AML cells, we examined the levels and capacity of the respiratory complexes in 1^o AML and normal PBSCs. When normalized for mitochondrial mass, 1^o AML cells (n = 12) had reduced activity of respiratory complexes III and IV compared to PBSCs (n = 10) (Mean complex III activity AML vs PBSC: 0.32 ± 0.04 RU vs 0.66 ± 0.11 RU p = 0.0063; Mean complex IV activity AML vs PBSC: 0.13 ± 0.01 RU vs 0.24 ± 0.02 RU, p= 0.0003). We evaluated the capacity of the respiratory complexes in AML cells and PBSCs by treating with increasing concentrations of the complex III inhibitor antimycin, and measuring the changes in oxygen consumption. AML cells displayed heightened sensitivity to the complex III inhibitor and less reserve capacity in the respiratory complex compared to PBSCs (mean concentration of antimycin required to reduce oxygen consumption by 50%: AML (n = 11) vs PBSC (n = 3): 13.7 ± 1.6 nM vs 29.0 ± 2.4 nM; p = 0.0007). AML cell lines were similar to 1^o AML cells with decreased basal respiratory complex activity and reserve capacity compared to PBSCs.

Given the reduced levels and reserve in the respiratory chain complexes in AML cells, we evaluated the effects of inhibiting mitochondrial protein translation in AML cells and PBSCs. Chemical (tigecycline, and chloramphenicol) and genetic (RNAi knockdown of the EF-Tu) inhibition of mitochondrial translation reduced the levels and function of the respiratory complexes that contain proteins encoded by mitochondrial DNA. Consistent with the reduced reserve capacity, inhibiting mitochondrial translation preferentially reduced oxygen consumption and viability of 1^o AML cells and AML cell lines over PBSCs and normal CD34+ cells.

To understand the molecular basis for the abnormal mitochondrial biogenesis in 1^o AML cells, we measured levels of the NRF-1, TFAM and EF-Tu, genes known to positively regulate mitochondrial biogenesis. Compared to PBSCs, AML samples showed at least a 3-fold increase in mRNA expression of these genes. Myc is a positive regulator of NRF-1, TFAM and EF-Tu. Therefore, we measured levels of myc in 1^o AML cells and PBSCs by Q-RT-PCR. Compared to PBSCs, myc was increased in 1^o AML cells and positively correlated with expression of NRF-1, TFAM and EF-Tu as well as with mitochondrial mass. To determine whether increased myc expression is functionally related to the increased mitochondrial biogenesis and decreased reserve in respiratory capacity, we employed P493 Burkitt's cells with inducible myc knockdown. P493 cells expressing myc had increased mitochondrial mass, larger mitochondria, and increased basal oxygen consumption compared to the myc knockdown cells. When normalized for mitochondrial mass, myc expressing cells had reduced activity of respiratory complexes III and IV compared to myc knockdown cells. In addition, myc expressing cells had less reserve in respiratory complex III (concentration of antimycin required to reduce oxygen consumption by 50% –+ myc P493 vs –myc P493: 6.580 ± 0.393 nM vs 12.87 ± 1.97 nM p =0.0352).

Thus, compared to normal hematopoietic cells, AML cells have greater mitochondrial mass but reduced reserve in their respiratory complexes. As a result of this decreased reserve, AML cells have a heightened sensitivity to inhibition of mitochondrial translation which reduces respiratory chain complex levels and activity. Genetically, the abnormal mitochondrial structure and function appears related to dysregulated myc and its influence on genes promoting increased mitochondrial biogenesis.