Metabolic control of BCR/Abl oncoprotein expression and maintenance of stem cell potential in Chronic Myeloid Leukaemia cells

Chronic Myeloid Leukaemia (CML) is a haematopoietic stem cell-driven myeloproliferative disease which occurs due to the reciprocal translocation between chromosomes 9 and 22, which gives birth to the Philadelphia chromosome (Ph). This chromosome bears the fusion oncogene BCR-ABL-1, encoding for the BCR/Abl oncoprotein, a constitutively active cytoplasmic tyrosine kinase. Ph+ leukaemic stem cells (LSC), like their normal counterpart, are hosted within specialized bone marrow (BM) sites named stem cell niches (SCN), where LSC fate is thoroughly controlled. Over the last two decades, with the development of tyrosine kinase inhibitors (TKi) such as Imatinib-Mesylate (Gleevec®), life expectancy of CML patients has improved significantly. Nevertheless, none of CML treatments, including that with TKi, is fully curative yet, as patients who have achieved complete cytogenetic response to therapy may still undergo relapse of disease. This is correlated to the long-term persistence of a very small subset of LSC capable to escape from therapy and thereby sustaining treatment-resistant minimal residual disease (MRD) of CML. In order to aim at cure, rather than care, of CML, it is therefore indispensable to design new therapeutic strategies to tackle that LSC subset. The project of this thesis was directed to deepen the role of SCN microenvironment in governing two crucial aspects of LSC, namely metabolism and quiescence, involved in CML cell ability to survive TKi treatment and thereby sustain MRD. To address these issues, three different BCR/Abl-positive stabilized cell lines (K562, KCL22, TF-1) have been used, as an experimental model ideal for biochemical studies. To investigate CML cell metabolism, we cultured cells under standard incubation conditions (atmosphere air/CO2) or in a gas-tight manipulator/incubator fuelled with a gas mixture containing 0.1% of oxygen, CO2 and the remaining of nitrogen, i.e. an atmosphere closely mimicking the metabolic scenario characteristic of SCN in vivo. Moreover, CML cell quiescence studies have been carried out by means of a human BM-like 3D model and the FUCCI system, a tool which enables to dynamically track cell cycle distribution. The results obtained led to establish that, in standard atmosphere, lactate is a powerful surrogate of glucose, able to sustain CML cell viability and growth, as well the maintenance of BCR/Abl oncoprotein. Furthermore, we demonstrated that it is actually lactate conversion to pyruvate, and the transport of the latter into the mitochondria where it fuels the TCA cycle, the pivotal player ensuring CML cell survival, growth and the maintenance of the expression of BCR/Abl oncoprotein. On the other hand, the experiments based on cell incubation under very low oxygen tension led to establish that such a metabolic pressure time-dependently forces CML cells to suppress BCR/Abl expression. Nevertheless, CML cells were found able to re-express the oncoprotein when transferred into growth-permissive secondary cultures incubated in air, underscoring the reversibility of BCR/Abl suppression. The downregulation of BCR/Abl in low oxygen was found tightly linked to glutamine-driven glucose consumption. As it was straightforward to correlate this consumption in low oxygen with lactate production, the role of the latter in low oxygen-incubated CML cells was investigated. Conversely to what previously demonstrated for air-incubated cells, lactate did not foster cell viability or enhanced BCR/Abl expression. Actually, extracellular lactate, by signalling via the GPR81 receptor, did exactly the opposite, indicating that a tissue environment heavily conditioned by glycolytic activity determines the suppression of CML oncoprotein and thereby refractoriness to TKi. CML cell quiescence, on the other hand, has been also indicated as one of the potential mechanisms sustaining MRD. In this respect, we found that the adherence to a human BM-like 3D structure enhances CML cell quiescence with respect to that of CML cells remaining in suspension. Thus, adherence to non-haematopoietic components of SCN may enhance CML cell refractoriness to TKi. Accordingly, TKi treatment blocked adherent CML cells in the G1 cell cycle phase but adherence sheltered cells from dying. On the basis of all above, the main general conclusion of the studies reported in this thesis is that the SCN microenvironment plays a fundamental role in sustaining treatment-resistant MRD of CML, as the refractoriness of CML cells to TKi is coupled not only to their ability to suppress the BCR/Abl oncoprotein as a consequence of their adaptation to the metabolic conditions of SCN, but also to their enhanced quiescence driven by the adherence to the SCN structure. A future perspective of this work is to acquire further knowledge of CML cell metabolism and quiescence within the SCN microenvironment, enabling to develop new therapeutic strategies capable to significantly reduce the LSC pool which sustains the MRD of CML.