The metabolic profile of Chronic Myeloid Leukaemia: stem cells as a target to overcome resistance to therapy.

Chronic Myeloid Leukaemia (CML) is a stem cell-driven disorder treated with Tyrosine Kinase inhibitors (TKi) with impressive efficacy. However, TKi are unable in most cases to prevent the relapse of disease, as even a very successful response to treatment results in the persistence of a state of Minimal Residual Disease (MRD). Our hypothesis predicts that MRD is sustained by the persistence of Leukaemic Stem Cells (LSC) capable to survive and cycle independently of BCR/Abl kinase activity within Bone Marrow (BM) stem cell niches where severe oxygen and glucose shortage would result in BCR/Ablprotein suppression. In this study, we addressed the role of the availability of glutamine, among a number of other metabolites possibly relevant in this context, in the control of BCR/Ablprotein expression in CML cell cultures where energy supply is markedly restricted, i.e. maintained under conditions likely mimicking those of stem cell niches in vivo. We found that glutamine drives accelerated BCR/Ablprotein suppression and that this phenomenon is paralleled by the kinetics of glucose consumption from culture medium. The relationship between presence of glutamine and glucose consumption was deepened by investigating the effects of different metabolic inhibitors. We found that the inhibition of glycolysis via treatment with 2-DG or 3PO, as well as that of Pentose Phosphate Pathway (PPP) via treatment with 6-AN, prevented the effects of the presence of glutamine on BCR/Ablprotein expression, confirming that BCR/Ablprotein suppression requires the presence of glutamine and depends on glucose consumption irrespective of the pathway driving this consumption. On the contrary, the inhibition of OxPhos by means of metformin did not interfere with the effects of the presence or absence of glutamine on BCR/Ablpr otein expression. The effects of the presence or the absence of glutamine were also tested on the maintenance of stem cell potential in low oxygen. Using our in vitro LSC assay, Culture-Repopulation Ability (CRA) assay, we found that cells grown in the absence of glutamine exploited their stem cell potential promptly upon transfer to permissive conditions. The treatment of glutamine-free cultures with metformin did not interfere with the pattern of LC2 repopulation. On the contrary, BCR/Ablprotein-negative cells were affected by metformin treatment. The treatment with BPTES, a GLS1 inhibitor, in either the presence or the absence of glutamine, favored the maintenance of BCR/Ablprotein expression in low oxygen, so that LSC transferred to permissive conditions were capable to exploit their stem cell potential rapidly, driving prompt LC2 repopulation. This result could represent the basis of an innovative CML treatment strategy using inhibitors of glutamine metabolism in combination with TKi to determine LSC eradication together with induction or maintenance of remission.