The Leukemic Stem Cell Niche: Adaptation to “Hypoxia” versus Oncogene Addiction

Previous studies based on low oxygen concentrations in the incubation atmosphere revealed that metabolic factors govern the maintenance of normal hematopoietic or leukemic stem cells (HSC and LSC). The physiological oxygen concentration in tissues ranges between 0.1 and 5.0%. Stem cell niches (SCN) are placed in tissue areas at the lower end of this range (“hypoxic” SCN), to which stem cells are metabolically adapted and where they are selectively hosted. The data reported here indicated that driver oncogenic proteins of several leukemias are suppressed following cell incubation at oxygen concentration compatible with SCN physiology. This suppression is likely to represent a key positive regulator of LSC survival and maintenance (self-renewal) within the SCN. On the other hand, LSC committed to differentiation, unable to stand suppression because of addiction to oncogenic signalling, would be unfit to home in SCN. The loss of oncogene addiction in SCN-adapted LSC has a consequence of crucial practical relevance: the refractoriness to inhibitors of the biological activity of oncogenic protein due to the lack of their molecular target. Thus, LSC hosted in SCN are suited to sustain the long-term maintenance of therapy-resistant minimal residual disease

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

uPAR Knockout Results in a Deep Glycolytic and OXPHOS Reprogramming in Melanoma and Colon Carcinoma Cell Lines

Urokinase Plasminogen Activator (uPA) Receptor (uPAR) is a well-known GPI-anchored three-domain membrane protein with pro-tumor roles largely shown in all the malignant tumors where it is over-expressed. Here we have exploited the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene knock out approach to investigate its role in the oxidative metabolism in human melanoma and colon cancer as the consequences of its irreversible loss. Knocking out PLAUR, a uPAR-encoding gene, in A375p, A375M6 and HCT116, which are two human melanoma and a colon carcinoma, respectively, we have observed an increased number of mitochondria in the two melanoma cell lines, while we evidenced an immature biogenesis of mitochondria in the colon carcinoma culture. Such biological diversity is, however, reflected in a significant enhancement of the mitochondrial spare respiratory capacity, fueled by an increased expression of GLS2, and in a decreased glycolysis paired with an increased secretion of lactate by all uPAR KO cells. We speculated that this discrepancy might be explained by an impaired ratio between LDHA and LDHB

Targeting the Extracellular Signal-Regulated Kinase 5 Pathway to Suppress Human Chronic Myeloid Leukemia Stem Cells

Summary: Tyrosine kinase inhibitors (TKi) are effective against chronic myeloid leukemia (CML), but their inefficacy on leukemia stem cells (LSCs) may lead to relapse. To identify new druggable targets alternative to BCR/ABL, we investigated the role of the MEK5/ERK5 pathway in LSC maintenance in low oxygen, a feature of bone marrow stem cell niches. We found that MEK5/ERK5 pathway inhibition reduced the growth of CML patient-derived cells and cell lines in vitro and the number of leukemic cells in vivo. Treatment in vitro of primary CML cells with MEK5/ERK5 inhibitors, but not TKi, strikingly reduced culture repopulation ability (CRA), serial colony formation ability, long-term culture-initiating cells (LTC-ICs), and CD26-expressing cells. Importantly, MEK5/ERK5 inhibition was effective on CML cells regardless of the presence or absence of imatinib, and did not reduce CRA or LTC-ICs of normal CD34+ cells. Thus, targeting MEK/ERK5 may represent an innovative therapeutic approach to suppress CML progenitor/stem cells. : Tyrosine kinase inhibitors targeting BCR/ABL are very effective against chronic myeloid leukemia (CML) cells but not leukemia stem cells (LSCs). Dello Sbarba, Rovida, and colleagues discovered that targeting the extracellular signal-regulated kinase 5 pathway in CML cell lines and primary cells results in the suppression of LSC as well as, especially in combination with tyrosine kinase inhibitors, the overall cell population. Keywords: leukemia stem cells, ERK5/MAPK, combination therapy, hypoxia, CML, MAPK7, MAP2K5, tyrosine kinase inhibitors/TKi, microenvironment, stem cell nich

Glutamine Availability Controls BCR/Abl Protein Expression and Functional Phenotype of Chronic Myeloid Leukemia Cells Endowed with Stem/Progenitor Cell Potential

International audienceThis study was directed to characterize the role of glutamine in the modulation of the response of chronic myeloid leukemia (CML) cells to low oxygen, a main condition of hematopoietic stem cell niches of bone marrow. Cells were incubated in atmosphere at 0.2% oxygen in the absence or the presence of glutamine. The absence of glutamine markedly delayed glucose consumption, which had previously been shown to drive the suppression of BCR/Abl oncoprotein (but not of the fusion oncogene BCR/abl) in low oxygen. Glutamine availability thus emerged as a key regulator of the balance between the pools of BCR/Abl protein-expressing and -negative CML cells endowed with stem/progenitor cell potential and capable to stand extremely low oxygen. These findings were confirmed by the effects of the inhibitors of glucose or glutamine metabolism. The BCR/Abl-negative cell phenotype is the best candidate to sustain the treatment-resistant minimal residual disease (MRD) of CML because these cells are devoid of the molecular target of the BCR/Abl-active tyrosine kinase inhibitors (TKi) used for CML therapy. Therefore, the treatments capable of interfering with glutamine action may result in the reduction in the BCR/Abl-negative cell subset sustaining MRD and in the concomitant rescue of the TKi sensitivity of CML stem cell potential. The data obtained with glutaminase inhibitors seem to confirm this perspective