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

This 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.

In Vitro Comparison of the Effects of Imatinib and Ponatinib on Chronic Myeloid Leukemia Progenitor/Stem Cell Features.

BACKGROUND: The development of molecularly tailored therapeutic agents such as the BCR/ABL-active tyrosine kinase inhibitors (TKi) resulted in an excellent treatment option for chronic myeloid leukemia (CML) patients. However, following TKi discontinuation, disease relapses in 40-60% of patients, an occurrence very likely due to the persistence of leukemic stem cells that are scarcely sensitive to TKi. Nevertheless, TKi are still the only current treatment option for CML patients. OBJECTIVE: The aim of this study was to compare the effects of TKi belonging to different generations, imatinib and ponatinib (first and third generation, respectively), on progenitor/stem cell expansion potential and markers. PATIENTS AND METHODS: We used stabilized CML cell lines (KCL22, K562 and LAMA-84 cells), taking advantage of the previous demonstration of ours that cell lines contain cell subsets endowed with progenitor/stem cell properties. Primary cells explanted from CML patients were also used. The effects of TKi on the expression of stem cell related genes were compared by quantitative PCR. Flow cytometry was performed to evaluate aldehyde-dehydrogenase (ALDH) activity and the expression of cluster of differentiation (CD) cell surface hematopoietic stem cell markers. Progenitor/stem cell potential was estimated by serial colony formation ability (CFA) assay. RESULTS: Ponatinib was more effective than imatinib for the reduction of cells with ALDH activity and progenitor/stem cell potential of CML patient-derived cells and cell lines. Furthermore, ponatinib was more effective than imatinib in reducing the percentage of CD26-expressing cells in primary CML cells, whereas imatinib and ponatinib showed similar efficacy on KCL22 cells. Both drugs strongly upregulated NANOG and SOX2 in CML cell lines, but in KCL22 cells this upregulation was significantly lower with ponatinib than with imatinib, an outcome compatible with a lower level of enrichment of the stem cell compartment upon ponatinib treatment. CONCLUSION: Ponatinib seems to target CML progenitor/stem cells better than imatinib.

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.

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.

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.