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Figure S6. Selective suppression of MIR300 pro-apoptotic but not anti-proliferative activity by TUG1 lncRNA in quiescent LSCs. A, BloodSpot array-based TUG1 expression levels during normal myelopoiesis and in myeloid neoplams. B, GEO Profiles show TUG1 levels in in lineage-negative (Lin-) and -positive (Lin+) CD34- and CD34human stem/progenitor cells from healthy individuals. C, Experimental data- and current literature-based graphic representation of signaling network controlling CML LSC quiescence and survival through the BMM-C/EBPbeta-MIR300 and BMM-TGFbeta-FoxM1 pathways. Dotted lines indicate inactive pathways, line thickness indicates relevance of the signal for LSC quiescence. Red lines indicate signals increasing MIR300 levels. Black lines signals increasing TUG1 levels. (bottom) effects of different TUG1 levels on CML leukemic stem (LSC) and progenitor (LPC) cell fate.

Figure S4. MIR300 anti-proliferative activity accounts for BMM-induced LSC entry into quiescence. A, Structure of 14q32 DLK1-DIO3 genomic imprinted locus hosting the MEG3-regulated human MIR300. B, MIR300 levels in 5-Aza- or DMSO-treated (24h) Ph+ cells. C, Effect of hypoxia on proliferation of CFSE+CD34+ CML-BC cells. D, left: Effect of MSC (HS-5)-derived CM on LAMA-84 proliferation expressed as fold changes of CFSE mean of fluorescence intensity (MFI)+/-SEM; middle: pro-apoptotic effect of PAD (FTY720; 2.5uM) and DMSO (control) on HS-5-cultured LAMA-84 cells; right: Effect of MSC (HS-5)-derived CM BCR-ABL1 expression (anti-ABL1) and activity anti-PY), phospho-BCR-ABL1, JAK2 expression and activity JAK2 Y1007/1008, PP2A activity (pPP2AY307 inactive form) and GRB2 used as a control (blots are representative of three independent experiments). E, Levels of C/EBPbeta and GRB2 mRNA and protein in HS-5 cells exposed to hypoxia (48h; 1% O2). F, Effect of neutralizing TGFbeta antibody (anti-TGFb Ab; 48h, 1.25 μg/ml) on MIR300 levels in CD34+ CML-BC cells. G, Effect of ectopic C/EBPalpha (MigR1-deltauORF-C/EBPalpha-HA) and C/EBPbeta (MigR1-C/EBPB-ERTAM) on MIR300 levels in K562 cells. Immunoblot shows levels of C/EBPbeta and GRB2 in normoxic and hypoxic K562 cells.

Range values of controls for the indicated experiments

Figure S3. MIR300 acts as master PP2A activator and inhibitor of G1/S transition. A, (left) Additional effects of MIR300 on SET, CCND2 and CDK6 expression; (right) KEGG/GO analysis of MIR300 effects on signal transduction pathways. B, CSmiRTar (filters: bone marrow normal and myeloid leukemia cells) and miRDIP-ComiR integrated analyses show functional clustering of predicted/validated MIR300 targets.

Figure S2. KEGG/GO analysis of MIR300 on PP2A-regulated signal transduction pathways. Cartoon shows the SET-dependent PP2A Inhibitory pathway in CML and the pleiotropic inhibitory effect of PP2A activation on validated and predicted MIR300 targets regulating G1/S cell cycle transition, Wnt-beta-catenin, TGFbeta, JAK-STAT, PI-3K-Akt, RAS-MAPK and Notch signaling pathways.

Figure S5. BMM-induced MIR300 anti-proliferative and PP2A-activating functions impair NK cell immune-response. A, PP2A-dependent regulation of MIR300 and miR-155 validated pathways predicted to occur also in the bone marrow endosteal niche. B, Effect of hypoxia (1% O2, 7 days) on CFSE+NK cell proliferation (% CFSE mean of fluorescence). C, HS-5 exosomal miRNAs reported as negative regulators of NK cell proliferation/activity and their experimentally validated targets. miRNA RNAseq was performed on an Illumina platform using libraries derived from 100 ng RNA/sample from HS-5 exosome purifications (n=3). D, Precursor (pre-miR-155) miR-155 (BIC) levels in resting and IL-12/IL-18 (18h)-stimulated NK cells exposed (48h) to HS-5 exosomes. E, Effect of hypoxia (1% O2) and HS-5 CM (48h) on TUG1 expression in CD56+CD3- primary NK and NK-92 cells. F, Effect of CpG-TUG1-shRNA and CpG-scramble (200-500 nM, 5 days) on IL-2-induced NK cell proliferation (% cell number). Data are represented as mean+/-SEM.

Figure S1. MIR300 activity in quiescent leukemic stem and progenitor cells. CFC-replating assays shows effects of lentiviral-mediated ectopic MIR300 expression, 250 nM and 500 nM CpG-miR-300 on serial replating activity (2nd replating) of leukemic chronic and acute CML and normal UCB CD34+CD38- HSC-enriched cell fractions. Infection with lentiviral empty vector and treatment with CpG-anti-MIR300 and CpG-scramble served as controls.

Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that

The fusion oncoprotein BCR-ABL1 exhibits aberrant tyrosine kinase activity and it has been proposed that it deregulates signaling networks involving both transcription factors and non-coding microRNAs that result in chronic myeloid leukemia (CML). Previously, microRNA expression profiling showed deregulated expression of miR-150 and miR-155 in CML. In this study, we placed these findings into the broader context of the MYC/miR-150/MYB/miR-155/PU.1 oncogenic network. We propose that up-regulated MYC and miR-155 in CD34+ leukemic stem and progenitor cells, in concert with BCR-ABL1, impair the molecular mechanisms of myeloid differentiation associated with low miR-150 and PU.1 levels. We revealed that MYC directly occupied the -11.7 kb and -0.35 kb regulatory regions in the MIR150 gene. MYC occupancy was markedly increased through BCR-ABL1 activity, causing inhibition of MIR150 gene expression in CML CD34+ and CD34- cells. Furthermore, we found an association between reduced miR-150 levels in CML blast cells and their resistance to tyrosine kinase inhibitors (TKIs). Although TKIs successfully disrupted BCR-ABL1 kinase activity in proliferating CML cells, this treatment did not efficiently target quiescent leukemic stem cells. The study presents new evidence regarding the MYC/miR-150/MYB/miR-155/PU.1 leukemic network established by aberrant BCR-ABL1 activity. The key connecting nodes of this network may serve as potential druggable targets to overcome resistance of CML stem and progenitor cells.

Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that MIR300 has antiproliferative and PP2A-activating functions that are dose dependently differentially induced by CCND2/CDK6 and SET inhibition, respectively. MIR300 is upregulated in CML LSCs and NK cells by bone marrow microenvironment (BMM) signals to induce quiescence and impair immune response, respectively. Conversely, BCR-ABL1 downregulates MIR300 in CML progenitors to prevent growth arrest and PP2A-mediated apoptosis. Quiescent LSCs escape apoptosis by upregulating TUG1 long noncoding RNA that uncouples and limits MIR300 function to cytostasis. Genetic and pharmacologic MIR300 modulation and/or PP2A-activating drug treatment restore NK cell activity, inhibit BMM-induced growth arrest, and selectively trigger LSC apoptosis in vitro and in patient-derived xenografts; hence, the importance of MIR300 and PP2A activity for CML development and therapy. Significance: Tumor-naïve microenvironment–induced MIR300 is the only tumor suppressor miRNA that induces CML LSC quiescence while inhibiting NK cell antitumor immune response, and CML LSC/progenitor cell apoptosis through its anti-proliferative and PP2A-activating functions, respectively. Thus, the importance of MIR300 and PP2A-activating drugs for formation/survival and eradication of drug-resistant CML LSCs, respectively. See related commentary by Broxmeyer, p. 13. This article is highlighted in the In This Issue feature, p. 5

Drug-resistance of tumor-initiating cells, impaired NK cell immune-response, PP2A loss-of-function and aberrant miRNA expression are cancer features resulting from microenvironmental- and tumor-specific signals. Here we report that genomic-imprintedMIR300is a cell context-independent dual function tumor suppressor which is upregulated in quiescent leukemic stem (LSC) and NK cells by microenvironmental signals to induce quiescence and impair immune-response, respectively, but inhibited in CML and AML proliferating blasts to prevent PP2A-induced apoptosis.MIR300anti-proliferative and PP2A-activating functions are differentially activated through dose-dependent CCND2/CDK6 and SET inhibition, respectively. LSCs escape PP2A-mediated apoptosis through TUG1 lncRNA that uncouples and limitsMIR300functions to cytostasis by regulating unbound-MIR300levels. HaltingMIR300homeostasis restores NK cell activity and suppresses leukemic but not normal hematopoiesis by eradicating nearly all LSCs. Thus,MIR300tumor suppressor activity is essential and therapeutically important for LSC-driven leukemias.

The increasing interest in exploring the human genome and identifying genetic risk factors contributing to the susceptibility to and outcome of diseases has supported the rapid development of genome-wide techniques. However, the large amount of obtained data requires extensive bioinformatics analysis. In this work, we established an approach combining amplified fragment length polymorphism (AFLP), AFLP in silico and next generation sequencing (NGS) methods to map the malignant genome of patients with chronic myeloid leukemia. We compared the unique DNA fingerprints of patients generated by the AFLP technique approach with those of healthy donors to identify AFLP markers associated with the disease and/or the response to treatment with imatinib, a tyrosine kinase inhibitor. Among the statistically significant AFLP markers selected for NGS analysis and virtual fingerprinting, we identified the sequences of three fragments in the region of DNA repeat element OldhAT1, LINE L1M7, LTR MER90, and satellite ALR/Alpha among repetitive elements, which may indicate a role of these non-coding repetitive sequences in hematological malignancy. SNPs leading to the presence/absence of these fragments were confirmed by Sanger sequencing. When evaluating the results of AFLP analysis for some fragments, we faced the frequently discussed size homoplasy, resulting in co-migration of non-identical AFLP fragments that may originate from an insertion/deletion, SNP, somatic mutation anywhere in the genome, or combination thereof. The AFLP–AFLP in silico–NGS procedure represents a smart alternative to microarrays and relatively expensive and bioinformatically challenging whole-genome sequencing to detect the association of variable regions of the human genome with diseases.

Leukemia emergence, maintenance, relapse and/or progression are causally linked to the presence of drug-resistant leukemia-initiating cells and impaired natural killer (NK) cell anti-tumor immune-response. Bone marrow microenvironment (BMM)- and/or leukemia-derived signals induce aberrant non-coding RNA expression and inhibit protein phosphatase 2A (PP2A) tumor suppressor activity. PP2A loss-of-function is essential for NK cell activity and leukemic but not normal stem and progenitor cell proliferation and survival. The human MIR300 gene is an intergenic miRNA that belongs to the 14q32.31 DLK1-DIO3 genomic-imprinted tumor suppressor miRNA cluster B. MIR300 was found involved in loss-of heterozygosity, inhibited in several tumor types with high mitotic index and during epithelial-to-mesenchymal transition (EMT), and associated with a cancer stem cell phenotype. By using primary cells from Philadelphia-positive (Ph+) chronic myelogenous leukemia (CML) in chronic (CP) and blastic (BC) phase, and complex karyotype (CK) acute myeloid leukemia (AML) patients, as paradigmatic examples of stem cell-derived neoplasms characterized by constitutive expression of oncogenic kinases, PP2A loss-of-function, altered microRNA expression and impaired NK cell proliferation and cytotoxicity, we found that MIR300 is a cell context-independent tumor suppressor with anti-proliferative and PP2A-dependent pro-apoptotic activities which are sequentially activated in a MIR300 dose-dependent manner through inhibition of CCND2/CDK6 and SET (PP2A inhibitor), respectively. To prevent PP2A-induced apoptosis, MIR300 is inhibited by oncogenic signals in CD34+CML (CP and BC) and CK-AML progenitors. Conversely, tumor-naïve BMM-induced C/EBPbeta-mediated signals (hypoxia and MSC exosomes) markedly upregulate MIR300 expression in primary CML and AML CD34+CFSEmax leukemic stem (LSC) and CD56+CD3-NK cells to induce/maintain quiescence (increased CD34+leukemic blasts in G0) and impair immune-response (suppression of NK cell proliferation and cytotoxic activity toward CD34+ leukemic blasts and CFSEmaxCD34+ CML-BC quiescent LSCs), respectively. Inhibition of MIR300 expression/activity rescues NK cell proliferation and anti-tumor cytotoxicity and prevented MSC- and hypoxia-induced growth-suppression of CD34+leukemic blasts by inhibiting degradation of MIR300 targets (e.g. SET, CCND2). We found that CML and AML LSCs escape MIR300-induced PP2A-mediated apoptosis through the hypoxia- and tumor-dependent TGFb1-FoxM1-mediated upregulation of TUG1 lncRNA. TUG1 is an oncogenic lncRNA described as a MIR300sponge and found upregulated in solid tumors, in which it has strong diagnostic, prognostic and therapeutic relevance and is associated with cancer stem cell maintenance and EMT. In quiescent CML and AML LSCs, TUG1 uncouples and limits MIR300 tumor suppressor functions to cytostasis by maintaining unbound MIR300 at levels sufficient to inhibit CCND2 and CDK6 but not SET expression. Exposure to clinically-relevant CpG-modified oligonucleotides modulating MIR300levels and/or inhibiting TUG1 MIR300-sponging activity, restores NK cell proliferation and cytotoxic activity, and suppresses human leukemic but not normal hematopoiesis by eradicating nearly all (> 95% reduction) CFSEmaxCD34+ and CD45+CD34+CD38-CD90+ LSCs and CD34+leukemic CML (CP and BC) and CK-AML blasts in vitro (CFCs, LTC-IC, and CFSEmaxCD34+cell tracking) and/or in NRG-SGM3 PDX mouse models of acute and chronic myeloid leukemias. Altogether, this work highlights the therapeutic importance of altering MIR300 expression in anti-LSC and NK cell-based approaches for myeloid leukemias, and indicates that tumor-naïve BMM-induced MIR300 tumor suppressor anti-proliferative and PP2A-activating functions may support leukemogenesis by promoting the formation and initial expansion of the quiescent LSC pool through the induction of LSC dormancy and inhibition of quiescent LSC killing by cytokine-activated NK cells, respectively. (G.S. and R.T. equally contributed to this work) Disclosures Stagno: BMS: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Deininger:TRM: Consultancy; Sangoma: Consultancy; Incyte: Honoraria; Novartis: Honoraria; Sangamo: Consultancy; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Humana: Honoraria; Ascentage Pharma: Consultancy, Honoraria; Blueprint: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Research Funding; Fusion Pharma: Consultancy; Adelphi: Consultancy. Milojkovic:BMS: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Apperley:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Baer:Takeda: Research Funding; Incyte: Research Funding; Kite: Research Funding; Forma: Research Funding; AI Therapeutics: Research Funding; Abbvie: Research Funding; Astellas: Research Funding.

MicroRNAs (miRNAs) constitute a robust regulatory network with post-transcriptional regulatory efficiency for almost one half of human coding genes, including oncogenes and tumour suppressors. We determined the expression profile of 667 miRNAs in colorectal cancer (CRC) tissues and paired non-tumoural tissues and identified 42 differentially expressed miRNAs. We chose miR-215, miR-375, miR-378, miR-422a and miR-135b for further validation on an independent cohort of 125 clinically characterized CRC patients and for in vitro analyses. MiR-215, miR-375, miR-378 and miR-422a were significantly decreased, whereas miR-135b was increased in CRC tumour tissues. Levels of miR-215 and miR-422a correlated with clinical stage. MiR-135b was associated with higher pre-operative serum levels of CEA and CA19-9. In vitro analyses showed that ectopic expression of miR-215 decreases viability and migration, increases apoptosis and promotes cell cycle arrest in DLD-1 and HCT-116 colon cancer cell lines. Similarly, overexpression of miR-375 and inhibition of miR-135b led to decreased viability. Finally, restoration of miR-378, miR-422a and miR-375 inhibited G1/S transition. These findings indicate that miR-378, miR-375, miR-422a and miR-215 play an important role in CRC as tumour suppressors, whereas miR-135b functions as an oncogene; both groups of miRNA contribute to CRC pathogenesis.

Inhibition of protein phosphatase 2A (PP2A) tumor suppressor is essential for chronic myelogenous leukemia (CML) stem cell (LSC) maintenance and disease development. Persistence of drug-resistant quiescent LSCs depends on cell-autonomous and bone marrow (BM) signals. Herein, we identified miR-300 as an miRNA inhibited in CD34+ CML progenitors and during blastic transformation (BC) through the BCR-ABL1-dependent inhibition of C/EBPβ-induced miR-300 transcription. In CML progenitors, ectopic mir-300 expression directly targets the PP2A inhibitory pathway (i.e., Jak2, hnRNPA1, SET) and other factors essential for LSC maintenance and disease progression (e.g., CCND1/2, b-catenin, Myc, Twist-1). In leukemic but not normal CD34+ cells, miR-300 acts as a potent tumor suppressor by inducing cell cycle exit and promoting apoptosis. Conversely, hypoxia-induced BCR-ABL1 inhibition and induction of C/EBPβ were found essential for increased miR-300 levels in quiescent LSCs, although mesenchymal stromal cells (MSC)-derived exosomal miR-300 also contributed to it. Moreover, low O2 levels and MSC-derived exosomes induced quiescence of CD34+ CML cells. Notably, expression of an anti-miR-300 in MSCs prevented exosome-induced CD34+ CML growth arrest. LSCs escaped miR-300-induced apoptosis through the autocrine/paracrine TGFβ1-induced expression of TUG1, a lncRNA acting as an miR-300 sponge. In fact, TUG1 or TGFβ1 inhibition decreased quiescent (CFSEMAX) LSC number. By contrast, miR-300 inhibition did not alter LSC survival/self-renewal, further supporting a role for TUG1 as an miR-300 sponge. Accordingly, TUG1 was markedly induced in CFSEMAX but not dividing CD34+ CML cells. In fact, low levels of ectopic miR-300 induced growth arrest (decreased LTC-IC and CFC/replating activity) without affecting quiescent LSC number. By contrast, high doses of miR-300 but not scramble CpG-ODN impaired LSC survival (LTC-IC) and self-renewal (CFC/replating), induced marked killing of quiescent LSCs and dividing progenitors, and impaired CML engraftment in NRG-SGM3 mice. Such effects were further enhanced when CPG-miR-300 and CPG-anti-TUG1 were combined. By contrast, high CpG-miR-300 levels did not affect normal CD34+ cell survival/self-renewal likely because of high TUG1 expression. Altogether our results indicate that while miR-300 loss is essential for survival/proliferation of leukemic progenitors, increased miR-300 levels are required for LSC maintenance. Thus, induction of TUG1 may occur to preserve LSC survival in the BM endosteal niche where quiescence is induced by MSCs and low O2 levels through abnormal miR-300 induction. Thus, disrupting the miR-300/TUG1 balance may represent a potential therapeutic approach for treatment/eradication of LSC-derived leukemias. This work is supported in part by NIH-NCI R01CA163800. Citation Format: Giovannino Silvestri, Lorenzo Stramucci, Justin Ellis, Jason Harb, Paolo Neviani, Bin Zhang, Klara Srutova, Gabriel Pineda, Catriona Jamieson, Bruno Calabretta, Fabio Stagno, Paolo Vigneri, Georgios Nteliopoulos, Philippa May, Alistar Reid, Ramiro Garzon, Denis-Claude Roy, Martin Guimond, Peter Hokland, Michael Deininger, Garrett Fitzgerald, Chris Harman, Francesco Dazzi, Dragana Milojkovic, Jane Apperley, Guido Marcucci, Jianfei Qi, Katerina Machova-Polakova, Xiaoxuan Fan, Maria Baer, Rossana Trotta, Danilo Perrotti. The tumor suppressor activity of miR-300 is detrimental for leukemia development but required for leukemia stem cell maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1134.

MiR-300 is a microRNA predicted to target multiple components of the BCR-ABL1/JAK2/hnRNPA1/SET/PP2A/β-catenin pathway, which is essential for survival/self-renewal of leukemic progenitors and quiescent TKI-resistant Ph+ hematopoietic stem cells (HSCs). Nanostring arrays analysis of bone marrow (BM) cells from healthy individuals (n = 5) and CML patients (n = 10) showed gradual inhibition of miR-300 expression (CML-CPmiR-300>CML-BCmiR-300). MiR-300 transduction in CMLCD34+ cells and BCR-ABL1+ cell lines decreased JAK2, β-catenin, hnRNPA1 and SET expression and increased PP2A activity. Targets were confirmed by miR-300 expression in BCR-ABL1+ cells expressing Flag-tagged miR-300-targets lacking or carrying a wild-type or mutated 3’UTR. Restored miR-300 expression in CMLCD34+ cells and/or BCR-ABL1+ cell lines impaired proliferation and clonogenic potential, markedly reduced LTC-ICs, and increased TKI sensitivity. Notably, miR-300 expression was inhibited by BCR-ABL1 in proliferating cells. Accordingly, imatinib restored miR-300 expression in CD34+ dividing progenitors and BCR-ABL1+ cell lines without altering miR-300 levels in quiescent (CFSEMAX) CMLCD34+ cells (n = 3), consistent with the BCR-ABL1 kinase-dependent activation of the Jak2/SET/PP2A/β-catenin pathway in CML progenitors but not quiescent Ph+ HSCs. Surprisingly, miR-300 levels were increased in CD34+CD38- compared to CD34+CD38+ CML cells, and >20-fold higher in CFSEMAX compared to dividing CMLCD34+ cells (n = 4). To determine whether enhanced miR-300 expression in quiescent cells depends on cell autonomous events or is induced by the BM microenvironment, we exposed BCR-ABL+ cells to conditioned medium (CM) of HS-5 or hTERT mesenchymal stem cells (MSC). CM strongly decreased proliferation, induced imatinib but not FTY720 (PP2A activator) resistance, increased miR-300 levels, decreased BCR-ABL1 activity and Jak2 expression but not its activity, and did not alter β-catenin levels or PP2A activity. Interestingly, miR-300 was found in MSC-derived exosomes, and its expression increased in BCR-ABL1+ cells exposed to exosomes. Accordingly, proliferation of CML-BCCD34+and LAMA-84 cells was strongly reduced upon exposure to MSC-derived exosomes. These effects were abolished when we used CM from MSCs transduced with a miR-300 antagomir. Altogether our results indicate that downregulation of miR-300 appears necessary for the activation of JAK2/SET/PP2A/β-catenin survival signals in CML progenitors. Conversely, increased miR-300 levels (endogenous and MSC-derived) seem to be required for HSC quiescence. Citation Format: Rossana Trotta, Giovannino Silvestri, Lorenzo Stramucci, Justin J. Ellis, Justine Yu, Jason J. Harb, Paolo Neviani, Guido Marcucci, Klara Srutova, Polakova K. Machova, Denis-Claude Roy, Peter Hokland, Michael Deininger, Ravi Bhatia, Carlo Gambacorti-Passerini, Dragana Milojkovic, Alistair Reid, Jane Apperley, Ferenc Livak, Jianfei Qi, Maria R. Baer, Danilo Perrotti. Role of the MSC-derived exosomal and endogenous JAK2-SET/PP2A-β-catenin-modulator miR-300 in leukemic stem/progenitor proliferation and survival in CML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 951.

MiR-300 is a microRNA predicted to target multiple components of the BCR-ABL1 / JAK2 / hnRNPA1 / SET / PP2A / β-catenin pathway, which is essential for survival/self-renewal of leukemic progenitors and quiescent TKI-resistant Ph+ hematopoietic stem cells (HSCs). Nanostring arrays analysis of bone marrow (BM) cells from healthy individuals (n=5) and CML patients (n=10) showed gradual inhibition of miR-300 expression (CML-CPmiR-300>CML-BCmiR-300). MiR-300 transduction in CMLCD34+ cells and BCR-ABL1+ cell lines decreased JAK2, β-catenin, hnRNPA1 and SET expression and increased PP2A activity. Targets were confirmed by miR-300 expression in BCR-ABL1+ cells expressing Flag-tagged miR-300-targets lacking or carrying a wild-type or mutated 3'UTR. Restored miR-300 expression in CMLCD34+ cells and/or BCR-ABL1+ cell lines impaired cell cycle progression, proliferation and clonogenic potential, markedly reduced LTC-ICs, and increased TKI sensitivity. Notably, miR-300 expression was inhibited by BCR-ABL1 in proliferating cells. Accordingly, imatinib restored miR-300 expression in CD34+ dividing progenitors and BCR-ABL1+ cell lines without altering miR-300 levels in quiescent (CFSEMAX) CMLCD34+ cells (n=3), consistent with the BCR-ABL1 kinase-dependent activation of the Jak2/SET/PP2A/β-catenin pathway in CML progenitors but not quiescent Ph+ HSCs. Indeed, ectopic SET expression counteracted the negative effects of mir-300 on cell proliferation and survival. Surprisingly, miR-300 levels were increased in CD34+CD38- compared to CD34+CD38+ CML cells, and >20-fold higher in CFSEMAX compared to dividing CMLCD34+ cells (n=4). To determine whether enhanced miR-300 expression in quiescent cells depends on cell autonomous events or is induced by the BM microenvironment, we exposed BCR-ABL+ cells to conditioned medium (CM) of HS-5 or hTERT mesenchymal stem cells (MSC). CM strongly decreased proliferation, induced imatinib but not FTY720 (PP2A activator) resistance, increased miR-300 levels, decreased BCR-ABL1 activity and Jak2 expression but not its activity, and did not alter b-catenin levels or PP2A activity. Interestingly, miR-300 was found in MSC-derived exosomes, and its expression increased in BCR-ABL1+ cells exposed to exosomes. Accordingly, proliferation of CML-BCCD34+ and LAMA-84 cells was strongly reduced upon exposure to MSC-derived exosomes. These effects were abolished when we used CM from MSCs transduced with a miR-300 antagomir. Altogether our results indicate that downregulation of miR-300 appears necessary for the activation of JAK2/SET/PP2A/b-catenin survival signals in CML progenitors. Conversely, increased miR-300 levels (endogenous and MSC-derived) seem to be required for HSC quiescence. Disclosures Deininger: Novartis: Other: Consulting or Advisory Role, Research Funding; BMS: Other: Consulting & Advisory Role, Research Funding; Celgene: Research Funding; Genzyme: Research Funding; Gilead: Research Funding; ARIAD Pharmaceutical Inc.: Other: Consulting or Advisory Role; Incyte: Other: Consulting or Advisory Role; Pfizer: Other: Consulting or Advisory Role. Milojkovic:BMS: Honoraria; ARIAD Pharmaceuticals Inc.: Honoraria; Novartis: Honoraria; Pfizer: Honoraria.

Introduction: Inhibition of the BCR-ABL protein activity by tyrosine kinase inhibitors (TKIs) plays crucial role in leukemic transformation from the chronic phase (CP) of the chronic myeloid leukemia (CML) into fatal blast crisis (BC). However 20 - 30 % of CML patients develop resistance to the TKIs. Beside mutations in BCR-ABL kinase domain, BCR-ABL independent mechanisms of acquired TKIs resistance including microRNAs (miR) may be also involved. A significant reduction of miR-150 levels associates with CML progression. MiR-150 is an inhibitor of the Myeloblastoma oncogene c-MYB, which is required for BCR-ABL-dependent leukemogenesis in a mouse model of CML-BC. Recently MYB was found to positively regulating another miR, miR-155, that inhibits tumor suppressor and pro-differentiation factor PU.1 in AML/MPS mouse model. PU.1 is a major regulator of myelopoiesis whose levels require precise guidance. We recently reported increased levels of miR-155 in CML-BC. Objectives: We hypothesize that BCR-ABL activity together with low levels of miR-150 activates putative oncogenic signaling pathway MYB/miR-155/PU.1 in CML leading to a progressive blockade of cell differentiation and possibly also of resistance to TKIs. Material and Method: Bone marrow (BM) cells of CML-CP patients at the time of diagnosis (n=10), and of treated patients either sensitive (n=8) or resistant (n=11) to TKIs, were FACS sorted for CD34 and CD38 into subpopulations representing the stages of cell differentiation. CD34+ and CD34- leukocytes from peripheral blood of healthy donors (n=10) were isolated by MACS and used as control. Levels of miR-150, BCR-ABL, MYB, miR-155 and PU.1 were determined by qRT-PCR. Expression of surface differentiation markers on BM cells was determined using flow cytometry at the time of diagnosis (n=10) and upon TKIs resistance (n=5). The effect of miR-150 overexpression and BCR-ABL inhibition was studied in CML-BC cell line K562 using nucleofection of the synthetic miR-150 and siRNA BCR-ABL (and by cell cultivation with TKI imatinib). mRNA and protein expression of BCR-ABL, MYB and miR-150 levels were determined by qRT-PCR and immunoblotting. Results: In the groups of healthy donors and de novo diagnosed CML patients we observed significant positive or negative correlations of expression levels between studied molecules among the sorted cell subpopulations, which outlined the activity of the putative pathway BCR-ABL/miR-150/MYB/miR-155/PU.1 in CML-CP. Interestingly, in the group of patients resistant to TKI, we found that correlation between MYB and miR-155 was not observed suggesting this pathway is deregulated upon TKI resistance. Conversely, we found gain of negative correlation between expression of MYB and PU.1 in the group of patients responding to TKI suggesting that MYB and PU.1 oppose each other upon normal cell differentiation. We also observed significant differences of the surface marker phenotype profiles between BM cells of de novo diagnosed CML patients and patients resistant to TKIs treatment. While relatively differentiated (granulocyte) CD34- CD38- CD11b+ CD14- population presents about 7 % of BM cells for the group of patients at diagnosis, it comprises only 0.3 % of BM cells for resistant group (P= 0.0027) indicating that TKI resistance associates with significant blockade of myeloid differentiation. We next utilized K562 cells showing that levels of miR-150 significantly increased after BCR-ABL inhibition by imatinib (P=0.0017) and also upon BCR-ABL knockdown with siRNA (P=0.002). This suggests that miR-150 is regulated by BCR-ABL. Similarly, MYB mRNA and protein levels were markedly decreased upon miR-150 overexpression and this was enhanced by inhibition of BCR-ABL activity with imatinib. Conclusions: Dysregulation of BCR-ABL/miR-150/MYB/miR-155/PU.1 pathway was observed in CML stages and stage-specific cell populations in relation to TKI-sensitivity and disease progression. Our model consists of the BCR-ABL upregulating MYB both through miR-150 inhibition and by other downstream mechanisms by additive effect, thus the cooperation of miR-150 and imatinib through inhibiting MYB may represent an important barrier to CML progression. Even more our results show that regulation of miR-155 and PU.1 may play role in resistance to TKI treatment. Supported by GAUK 178215 and by project 00023736 from the Czech Ministry of Health Disclosures Klamova: Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Machova Polakova:Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.