Inhibition of protein phosphatase 2A (PP2A) tumor suppressor is essential for chronic myelogenous leukemia (CML) stem cell (LSC) maintenance, disease evolution and innate anti-cancer immunity. In CML, persistence of ABL tyrosine kinase inhibitor (TKI)-resistant quiescent LSCs, disease development and inhibition of natural killer (NK) cell growth and anti-cancer activity depend on cell-autonomous and bone marrow (BM)-generated signals.Here we show that expression profiling of CD34+ BM progenitors from healthy individuals and CML patients in chronic (CP) and blastic (BC) phase identified a discrete number of microRNA (miRs) altered in CML and/or during blastic transformation. Among the miRs downregulated in CML, we focused on miR-300 because it directly inhibited expression of multiple components of the PP2A inhibitory pathway (i.e. Jak2, hnRNPA1, SET) and of other factors essential for LSC maintenance and disease progression (e.g. CCND1/2, b-catenin, Myc, Twist-1), when ectopically expressed in CML but not in normal CD34+ cells.MiR-300 acted as a potent tumor suppressor in leukemic CD34+ progenitors by inducing cell cycle exit and promoting spontaneous and (TKI)-induced apoptosis, whereas no effects were exerted in normal miR-300-transduced CD34+ cells. In CD34+ CML-BC progenitors miR-300 downregulation required the BCR-ABL1 kinase-dependent inhibition of C/EBPb, which was capable of binding and transcriptionally activating miR-300 promoter.Conversely, low O2 levels and mesenchymal stromal cell (MSC)-derived exosomes abnormally increased miR-300 expression in a TKI-insensitive manner to induce and/or preserve LSC quiescence and suppress NK cell growth. Indeed, lentiviral-mediated miR-300 inhibition in MSCs significantly prevented the MSC growth inhibitory activity on CD34+ CML-BC and NK cells. Accordingly, miR-300 levels were found higher in CD56+CD3- NK cells from CML patients at diagnosis compared to healthy individuals.Induction/maintenance of high miR-300 levels in LSCs (CD34+ CFSEMAX and CD34+CD38- cells) required the hypoxia-induced inhibition of BCR-ABL1 activity and induction of C/EBPb expression/activity. However, MSC-derived exosomal miR-300 also contributed to increase miR-300 expression in LSCs.LSCs escaped from the miR-300 pro-apoptotic activity through an autocrine/paracrine mechanism that required the release by CD34+ CML stem/progenitors of TGFb1 that, in turn, induced the expression of TUG1, a lncRNA acting as a miR-300 sponge. In fact, shRNA-mediated TUG1 suppression or Ab-dependent TGFb1 inhibition decreased the quiescent (CFSEMAX) CD34+ LSC number. By contrast, miR-300 inhibition did not alter LSC survival and self-renewal, further supporting a role for TUG1 as a miR-300 sponge. Accordingly, TUG1 was markedly induced in CFSEMAX but not dividing CD34+ CML cells. In fact, low ectopic miR-300 expression induced growth arrest (decreased LTC-ICs and CD34+CD38- CFC/replating activities) without affecting the number of CFSEMAX LSCs.By contrast, high doses of miR-300 (CPG-miR-300; 500nM) but not scramble oligonucleotides impaired LSC survival (LTC-IC) and self-renewal (CFC/replating), induced a marked killing of quiescent LSCs (decreased CD34+ CFSEMAX cells) and dividing CD34+ CML progenitors, and impaired CML-BC engraftment in NRG-SGM3 mice. Notably, the association of high CPG-miR-300 with TUG1 silencing (CPG-anti-TUG1) further reduced CD34+ CFSEMAX LSC number. Notably, TUG1 was highly expressed in normal CD34+ stem/progenitor cells; in fact, neither low nor high miR-300 levels altered their survival and/or self-renewal.Altogether our results indicate that while loss of miR-300 expression is essential for survival/proliferation of leukemic progenitors and, therefore, for development of a full-blown leukemia, increased miR-300 expression is necessary for reduced NK cell number/activity and maintenance of the LSC reservoir. In this scenario, induction of TUG1 may occur to preserve LSC survival after migration into the BM endosteal niche where quiescence is induced by MSCs and low O2 levels through marked induction of intracellular (endogenous and exosomal) miR-300 levels. Thus, disrupting the miR-300/TUG1 balance may represent a potential therapeutic approach for treatment/eradication of LSC-derived leukemias and restoration of innate immunity.This work is supported in part by NHI-NCI R01CA163800-01 DisclosuresGambacorti-Passerini:Pfizer: Consultancy, Honoraria, Research Funding; BMS: Consultancy. Deininger:ARIAD: Consultancy; Incyte: Consultancy; BMS: Consultancy, Research Funding; Celgene: Research Funding; Pfizer: Consultancy; Novartis: Consultancy, Research Funding; Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding. Milojkovic:ARIAD: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Honoraria, Speakers Bureau. Apperley:Pfizer: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Therakos: Honoraria; Incyte: Honoraria; Sun Pharma: Honoraria; Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses , Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding.