Mitochondrial structural features can not only modify the ability to generate ATP by mitochondrial oxidative phosphorylation but can also commit apoptosis, which could be compromised leading to chemoresistance. Knowing the critical role of respiratory capacity in AML1, it would not be surprising that ultrastructural disorders of mitochondria could be implicated in AML pathogenesis3,14. In this context, we investigated the mitochondrial ultrastructure in five leukemic cell lines that represent the different subtypes of AML together with their functional capacity.
Firstly, we showed that there were different respiratory profiles in basal and under stressful conditions according to leukemia cell lines subtypes. On one hand, we identified a subgroup of “low respiration” leukemia cell lines, including KG1 and OCI-AML3. Their percentage of spare respiratory capacity and maximal respiration was significantly reduced compared to the other subgroups of cell lines that could be considered as a “high respiration” leukemia cell line, including K562, HEL, HL60. KG1 showed the lowest basal respiration, the lowest maximal respiration, the lowest ATP production and the lowest spare respiratory capacity % compared to K562, HEL, HL60. This result was concordant with previous OCR studies concerning KG1 as low OXPHO cell lines15. Furthermore, we observed alterations of ROS production that were correlated to the respiratory profiles of leukemic cells. KG1 was less able to produce ROS in basal conditions and conditions stimulating mitochondrial. This first set of results emphasizes that leukemic cells display various alterations of their mitochondrial function which are probably linked to their molecular status. Indeed, NGS analysis of the five cell lines revealed that molecular alterations are significantly different between cell lines.
Our ultrastructural analysis of mitochondria firstly highlighted the prominent importance of the number of mitochondria on the respiratory profiles. Indeed, we showed that KG1 presented the lowest number of mitochondria/cells which could explain its bioenergetics characteristics. On the contrary, K562, which has the highest basal respiration, the highest maximal respiration and the spare respiratory capacity compared to the other leukemia cell lines, displayed the higher number of mitochondria per cell. Similarly, both HL60 and HEL presented a higher number of mitochondria per cells and higher maximal respiration than KG1. K562 and HL60 also presented the highest ROS emission regardless of the experimental conditions. Interestingly, K562 harbors an ASXL1 mutation that has been shown to enhance mitochondrial activity and to elevate ROS levels in a mouse model16. Therefore, we could hypothesize that ASXL1 mutation could, at least partially, induce as “high respiration” profile through alteration of mitochondrial number.
Since the enzymes involved in oxidative phosphorylation are located on the cristae and the cristae morphology adapts to energy requirements in normal cells17,18, we focus on the number of cristae and cristae diameter in leukemia cell lines. K562, HEL and OCI-AML3 presented the higher OCR consumption during ATP production compared to KG1, and as expected, HEL and OCI-AML3 harbored a high number of cristae per mitochondrion. However, K562 presented few cristae per mitochondrion even its high ATP production level. Taking into account its high number of mitochondria, it suggests that leukemic cells could regulate their number of mitochondria to maintain high respiratory levels despite structural defects. Moreover, we showed that HEL, OCI-AML3 and K562 carry high cristae diameter compared to KG1, to a less level to HL60 with the highest levels of ATP production. That is concordant with previous reports showing that the cristae diameter also gives information about mitochondria’s capacity to generate ATP19. In synthesis, the mitochondria content measured by the number of mitochondria per cell seems to drive the basal and maximal respiration of leukemia cells and, by consequence, the spare respiratory capacity (%), whereas the cristae diameter seems to better reflect the ATP production. On the opposite, the number of cristae appears as a versatile indicator of ATP production.
Analysis of mitochondria by electron microscopy also depicted disparity in the presence of mitochondrial-derived vesicle (MDV) precursors. Originally, MDVs were described in the mitophagy process and enabled the mitochondria to repair damages and restore their normal function9. The mitophagy is essential for the regulation of mitochondria degradation and efficiency. Autophagy and mitophagy are mostly described in myelodysplastic disorders. The budding formation of mitochondria’s outer membrane – the first step of MDVs production - was less frequent in K562 than in the other cell lines. Mitophagy deficiency of K562 could lead to the accumulation of damaged mitochondria, explaining the high number of mitochondria/cells in K562 and the “messy” appearance of K562s’ cristae. Recently, the induction of mitophagy in AML appears interesting as a novel cell death mechanism especially in FLT3-ITD subgroups, that also arbored mitophagy deficiency20. Knowing that the ASXL1 mutant leukemia cell line is also mitophagy-deficiency, it could be interesting to test the induction-mitophagy strategy not only in FLT3-ITD subgroups but also in ASXL1 mutant.
Interestingly, the ultrastructural analysis also gives information about calcium homeostasis through analysis of matrix granules, whose role in calcium sequestration was suggested, as their size and density increased when a high concentration of calcium was present in the fluid21. Interestingly, these matrix granules could also regulate the internal ionic mitochondrial environment13 and, thus, impact mitochondrial respiration. In our study, the presence of matrix granules differed according to the cell lines, although they were simultaneously cultured, fixed or colored. The HL60 cell line - an in vitro model of AML2, carrying the amplification of the proto-oncogene MYC - displayed many matrix granules. On the contrary, none of them were present in the K562 cells that express the BCR-ABL transcript. This observation could be concordant with an indirect role of the matrix granules in the calcium homeostasis. Indeed, the bcr-abl oncogene was shown to reduce the endoplasmic reticulum releasable calcium levels22, while MYC expression -a calcium-dependent proto-oncogene- increased intracellular calcium concentration23. Then, we described the presence of the close association of the endoplasmic reticulum with the mitochondria outer membrane known as “Mitochondria-outer mitochondrial membrane that also plays a role in the modulation of the Ca2 + homeostasis in the cancer cells10. In fact, the proximity of the mitochondrial and rough endoplasmic reticulum makes selective Ca2 + uptake possible by the mitochondria24. In our study, these structures could be observed in all the leukemia cell lines, confirming that this modification exists in AML. Interestingly, they were more abundant in HL60 than in K562 cells, where RER was less extensive. Recently, targeting the endoplasmic reticulum-mitochondria interface appears as a novel strategy to make leukemia more sensitive to cytostatics11 by releasing calcium from the endoplasmic reticulum and inducing apoptosis. Our work suggests that this strategy must be personalized to calcium-dependent leukemia having, for instance, a huge amount of matrix granules.