Activation of PP2A by FTY720 Inhibits Survival and Self-Renewal of the Ph(+) Chronic Myelogenous Leukemia (CML) CD34⁺/CD38⁻ Stem Cell through the Simultaneous Suppression of BCR/ABL and BCR/ABL– independent Signals

CML is a clonal disorder of the pluripotent hematopoietic stem cell characterized by the sustained kinase activity of the BCR/ABL oncoprotein. We reported that the BCR/ABL-dependent and SET-mediated inhibition of protein phosphatase PP2A tumor suppressor activity is essential for the leukemogenic potential of CD34⁺ CML bone marrow progenitors, as molecular and pharmacologic restoration of PP2A inhibits the activity of BCR/ABL and that of several important regulators of cell survival/proliferation, thus resulting in marked apoptosis, impaired clonogenic potential and in vivo leukemogenesis of imatinib/dasatinib-sensitive and -resistant Ph(+), but not normal, CD34⁺ blasts and/or BCR/ABL⁺ mouse marrow progenitors. Here we show that SET-dependent suppression of PP2A activity is a common feature of Ph(+) progenitors (CMP and GMP) and imatinib/ dasatinib-insensitive CD34+/CD38- BCR/ABL⁺ (n=3) stem cells but not of the equivalent cell fractions from healthy individuals (n=3). To determine the biological importance and therapeutic implications of impaired PP2A activity in Ph(+) stem cells, we evaluated by clonogenic, CFC/replating, LTC-IC and CFSE-mediated cell division-tracking assays, the effects of FTY720 (2.5 mM), a PP2A activator currently in phase III trials for MS patients, and lentiviral-mediated ectopic PP2Ac expression on survival and self-renewal of BCR/ ABL⁺ stem/progenitor cells isolated from bone marrow of CML blast crisis patients (n_tot=8; Ph1≥90%) and/or SCL-tTA-BCR/ABL transgenic animals (n_tot=10). FTY720 treatment (2.5–5mM) severely suppressed the clonogenic potential of CD34⁺/CD38⁻ and CD34⁺/CD38⁺/CD45RA^−/+ CML stem/progenitor cells. Accordingly, self-renewal and long-term repopulating potential of CML leukemic stem cells was markedly impaired by pharmacologic PP2A reactivation. In fact, the CFC output of LTC-IC cultures (6 weeks) deriving from FTY720-treated (2.5 mM; 72h) Ph(+) CD34⁺ cells was more than 95% inhibited if compared to that of LTC-IC cultures from untreated CML cells. By contrast, imatinib (5 mM) and dasatinib (200 nM) treatment led to a 3.5 and 5-fold increase in CFC output, respectively. Consistent with the ability of FTY720 to impair self-renewal of CML stem cells, a 50–90% reduction of the CFSE^MAX/quiescent cell population was observed in CFSE-stained CD34⁺ CML cells treated for 6–9 days with FTY720. Notably, FTY720 did not exert any significant effect on CFSE-stained CD34⁺ cells from healthy individuals whereas, as expected, imatinib (5 mM) and dasatinib (200 nM) treatment led to a 22% and 27% increase in CFSE^MAX CML cells, respectively. Interestingly, only FTY720 triggered apoptosis of CFSE^MAX CML cells (41% Annexin V⁺ cells) although BCR/ABL activity (phospho-ABL intracellular flow-cytometry staining) in CFSE^MAX cells was efficiently inhibited by FTY720, Imatinib and dasatinib, suggesting that BCR/ABL-independent PP2A-regulated signals control the survival and self-renewal of CML stem cells. Indeed, lentiviral-driven PP2Ac-overexpression as well as treatment with FTY720, but not imatinib, significantly decreased (40–90% reduction) CFC/serial replating efficiency, colony size and percentage of CFSE^MAX fraction (66–96% reduction) of Lin⁻/ Sca⁺/Kit⁺ (LSK) cells isolated from bone marrow and spleen of leukemic SCL-tTA-BCR/ ABL mice. Mechanistically, the detrimental effect of PP2A activation on survival and self-renewal of CML stem cells might depend on the ability of PP2A to inactivate b-catenin that, reportedly, is a PP2A target essential for the self-renewal of the CML blast crisis GMP progenitors. In fact, immunoblotting, direct immunofluorescence and LET/TCF luciferase assays showed that ectopic PP2Ac expression and/or FTY720, but not imatinib, treatment leads to inactivation/degradation of nuclear b-catenin in BCR/ABL⁺ primary mouse LSK and/or 32D-BCR/ABL cells. Altogether our data not only highlight the importance of PP2A inactivation for survival and self-renewal of CML stem cells but also suggest the existence of BCR/ABL-independent, PP2A-sensitive and b-catenin-mediated signals that may account for resistance of CML quiescent stem cells to tyrosine kinase inhibitor monotherapy. Thus, FTY720 treatment has the potential to eradicate CML by efficiently targeting both stem and progenitor Ph(+) cells regardless of their degree of sensitivity to imatinib and dasatinib.