Contenido mínimo de la ley de extinción de dominio

In this chapter, we first showed that primitive CD34+ _{CML cells enhance FAO in}

comparison to CD34- _{cells. Interestingly, FAO has been shown to play a crucial role}

in HSCs maintenance by producing cytosolic NADPH that prevents high ROS levels (151). Similarly, LSCs in AML have been suggested to rely on FAO for their survival (226). The selective increase in PC but not in PDH activity observed in CD34+ _CML

cells supported their FAO profile, as elevated acetyl coA levels from FAO are expected to increase PC activity. Moreover, we demonstrated that mitochondrial respiration and the levels of 13_{C-labelled isotopologues of TCA cycle metabolites}

from 13_{C16-palmitate and} 13_{C6-glucose were significantly increased in CD34}+ _CML

cells in comparison to CD34- _{CML cells.}

Altogether, these results have demonstrated that CD34+ _{CML cells display a}

significant increase in oxidative metabolism in comparison to CD34- _{CML cells.}

Previous studies have shown that normal HSCs rely primarily on glycolysis for their energy production and possess low mitochondrial functions (140, 146). Therefore, it would have been interesting to compare the metabolism of CD34+ _{and CD34}-

normal cells to see whether a similar metabolic difference is observed in normal haematopoietic cells. These experiments were however not performed due to lack of primary material. Moreover, the cellular composition of differentiated cells varies between normal and leukaemic haematopoiesis - the latter being characterised by an expansion of the myeloid cell lineage, which prevents a direct comparison between normal and leukaemic differentiated cells. One possibility would have been to sort for different cell types and analyse their metabolism individually. However, this study did not aim to assess the metabolic differences between HSC and progenitors at homeostasis and future investigations are necessary to address this point.

Our metabolic comparison of CD34+ _{cells from CML patients and normal cell donors}

revealed that FAO is elevated in CD34+ _{CML cells compared to CD34}+ _{normal cells.}

Accordingly, CD34+ _{CML cells had a significant increase in palmitate-derived TCA}

cycle intermediates and derived amino acids, confirming that the aforementioned increase in FAO in primitive CML cells is selective to them. Moreover, we have demonstrated that mitochondrial respiration, glucose oxidation and anaplerosis

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were significantly elevated in CD34+ _{leukaemic cells compared to normal}

counterparts. Noteworthy, a significant difference regarding the proliferation status of CD34+ _{CML and normal cells exists, with BCR-ABL driving the proliferation}

of CD34+ _{CML cells and CD34}+ _{normal cells being mainly quiescent in our in vitro}

culture conditions. Therefore, proliferating CD34+ _{CML cells and quiescent CD34}+

normal cells are expected to have distinct metabolic requirements and it could be argued that an increase in metabolic activity, such as the higher OXPHOS observed in CD34+ _{CML cells, was somewhat predicted. Conversely, studies have described}

that highly proliferative cancer cells tend to rely mainly on glycolysis for their energy production; albeit more recent studies have shown that mitochondrial oxidative metabolism is crucial for cancer progression and maintenance (1.5.3 and 1.5.4). To circumvent the proliferation bias, we assessed the ratio between the oxidative and glycolytic pathway in CD34+ _{CML and normal cells by measuring}

respectively the contribution of glucose-derived carbons into glutamate and lactate (Fig. 3.10). The ratio of glucose-derived glutamate (13_C

≥2-Glutamate) over

glucose-derived lactate (13_{C3-Lactate) was significantly higher in CD34}+ _{CML cells}

in comparison to CD34+ _{normal cells, confirming that oxidative metabolism is}

significantly elevated in primitive leukaemic cells despite normalising for the intrinsic characteristics of the cells.

These experiments were performed on CD34+ _{cells, consisting of a mixture of}

progenitors and stem cells. There are currently more rigorously-defined HSC and LSC populations and using these defined subsets of cells might have been more appropriate in this study. We therefore decided to recapitulate some of our key experiments in more stem cell-enriched subsets (CD34+_CD38-_{). To our knowledge,}

this was the first time that targeted metabolomics in this rare and primitive haematopoietic cell population was performed from patient-derived material. Of note, these experiments were only done in two patients and two normal cell donors given the limitations of working with very small cell populations from primary material. Indeed, to isolate CD34+_CD38- _{cells, CML and normal}

haematopoietic cells are first selected for expression of the CD34 cell surface marker, which represents about 1% of total leukocytes. Then, 20 millions of CD34+

cells are subjected to a FACS for selection CD34+_CD38- _{cells, which yields about}

0.5-1 million cells. We therefore optimised all our techniques to measure intracellular metabolites in a few number of suspension cells, and despite these

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technical difficulties, we managed to perform targeted metabolomic analysis in CD34+_CD38- _{cells from two CML patients and two normal donors. The data obtained}

from the two independent experiments were very similar and in line with our previous results, confirming that primitive CML cells possess increase mitochondrial functions in comparison to normal counterparts. The comparison of the metabolic phenotype between CD34+_CD38- _{and CD34}+_CD38+ _{cells indicated an}

interesting difference between normal and CML cells. While in leukaemic cells, the oxidative metabolism was similar in CD34+_CD38- _{and CD34}+_CD38+ _{cell subsets,}

our results indicated that CD34+_CD38- _{normal cells were more glycolytic than}

CD34+_CD38+ _{normal cells, in line with previous studies demonstrating that normal}

HSCs display low mitochondrial metabolism in comparison to progenitor cells. Of note, more primitive markers are available to further enrich for stem cells, but this was not possible given the number of cells required in our assays. As such, primitive HSCs are characterised phenotypically by being lineage (Lin) negative CD34+_CD38−_CD90+_CD45RA− _{(237). Future technological developments are required}

to investigate the metabolism of this rare population and address the metabolic difference between CD34+_CD38−_CD90+_CD45RA− _{normal and CML cells.}

Figure 3. 10 CD34+ _{CML cells display increased glucose oxidation compared to normal}

counterparts.

Ration of glucose-derived glutamate (13_C

2-5 Glutamate) over glucose-derived lactate (13C3 lactate) in CD34+ _{CML and normal cells measured by LC-MS following 24 h incubation with} 13_C

6-labelled glucose. Mean ± S.E.M. n=5 patients and normal samples. P value was calculated by unpaired Student’s t-test.

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