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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.

Hematopoiesis is a complex process that is regulated at multiple levels and through both intrinsic and extrinsic regulators. Trim28 is a multidomain protein that functions as a transcription factor and E3 ubiquitin ligase. Recent studies have established the pivotal roles of Trim28 in a variety of pathophysiologic processes including inflammation, autoimmune disorders, viral pathogenesis, cancer, tumor microenvironment and epithelial-to- mesenchymal transition. However, importance of Trim28 in early hematopoiesis, particularly in the maintenance and functions of hematopoietic stem and progenitor cells (HSPCs), remains largely unknown. In the present study, we identified Trim28 as a critical regulator of self-renewal, quiescence, and functions of hematopoietic stem cells (HSC). Conditional ablation of Trim28 in HSCs leads to perinatal lethality, due to pancytopenia within the hematopoietic lineage. Loss of Trim28 in HSCs affects their differentiation into myeloid-, erythroid-, and lymphoid- lineages. Trim28 deletion leads to altered frequencies and absolute numbers of HSCs, Multipotent Progenitors (MPPs) and lineage committed progenitors in the bone marrow (BM). Primary- and secondary- BM transplantation studies identified an indispensable and a cell-intrinsic role for Trim28 in HSCs. Mechanistic studies identified hyperproliferation of HSPCs and deregulated expression of cell cycle regulators in HSPCs. Strikingly, gene expression studies demonstrated that Trim28 deficiency impairs the transcriptional landscape and transcription factors signatures of HSPCs. In essence, these studies highlight a previously unknown role for Trim28 in the maintenance, functions, and multi-lineage differentiation of HSPCs.

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is primarily responsible for coronavirus disease (COVID-19) and it is characterized by respiratory illness with fever and dyspnea. Severe vascular problems and several other manifestations, including neurological ones, have also been frequently reported, particularly in the great majority of “long hauler” patients. SARS-CoV-2 infects and replicates in lung epithelial cells, while dysfunction of endothelial and neuronal brain cells has been observed in the absence of productive infection. It has been shown that the Spike protein can interact with specific cellular receptors, supporting both viral entry and cellular dysfunction. It is thus clear that understanding how and when these receptors are regulated, as well as how much they are expressed would help in unveiling the multifaceted aspects of this disease. Here, we show that SH-SY5Y neuroblastoma cells express three important cellular surface molecules that interact with the Spike protein, namely ACE2, TMPRSS2, and NRP1. Their levels increase when cells are treated with retinoic acid (RA), a commonly used agent known to promote differentiation. This increase matched the higher levels of receptors observed on HUVEC (primary human umbilical vein endothelial cells). We also show by confocal imaging that replication-defective pseudoviruses carrying the SARS-CoV-2 Spike protein can infect differentiated and undifferentiated SH-SY5Y, and HUVEC cells, although with different efficiencies. Neuronal cells and endothelial cells are potential targets for SARS-CoV-2 infection and the interaction of the Spike viral protein with these cells may cause their dysregulation. Characterizing RNA and protein expression tempo, mode, and levels of different SARS-CoV-2 receptors on both cell subpopulations may have clinical relevance for the diagnosis and treatment of COVID-19-infected subjects, including long hauler patients with neurological manifestations.

Peer review is the driving force of journal development, and reviewers are gatekeepers who ensure that Healthcare maintains its standards for the high quality of its published papers [...]

Peer review is the driving force of journal development, and reviewers are gatekeepers who ensure that Pharmaceuticals maintains its standards for the high quality of its published papers [...]

Peer review is the driving force of journal development, and reviewers are gatekeepers who ensure that Genes maintains its standards for the high quality of its published papers [...]

Peer review is the driving force of journal development, and reviewers are gatekeepers who ensure that Cancers maintains its standards for the high quality of its published papers [...]

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

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

The use of imatinib, second and third generation ABL tyrosine kinase inhibitors (TKI) (i.e. dasatinib, nilotinib, bosutinib and ponatinib) made CML a clinically manageable and, in a small percentage of cases, a cured disease. TKI therapy also turned CML blastic transformation into a rare event; however, disease progression still occurs in those patients who are refractory, not compliant with TKI therapy or develop resistance to multiple TKIs. In the past few years, it became clear that the BCR-ABL1 oncogene does not operate alone to drive disease emergence, maintenance and progression. Indeed, it seems that bone marrow (BM) microenvironment-generated signals and cell autonomous BCR-ABL1 kinase-independent genetic and epigenetic alterations all contribute to: i. persistence of a quiescent leukemic stem cell (LSC) reservoir, ii. innate or acquired resistance to TKIs, and iii. progression into the fatal blast crisis stage. Herein, we review the intricate leukemic network in which aberrant, but finely tuned, survival, mitogenic and self-renewal signals are generated by leukemic progenitors, stromal cells, immune cells and metabolic microenvironmental conditions (e.g. hypoxia) to promote LSC maintenance and blastic transformation.

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.

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.

The use of tyrosine kinase inhibitors (TKI), including nilotinib, has revolutionized the treatment of chronic myeloid leukemia (CML). However current unmet clinical needs include combating activation of additional survival signaling pathways in persistent leukemia stem cells after long-term TKI therapy. A ubiquitous signaling alteration in cancer, including CML, is activation of reactive oxygen species (ROS) signaling, which may potentiate stem cell activity and mediate resistance to both conventional chemotherapy and targeted inhibitors. We have developed a novel nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, imipramine blue (IB) that targets ROS generation. ROS levels are known to be elevated in CML with respect to normal hematopoietic stem/progenitor cells and not corrected by TKI. We demonstrate that IB has additive benefit with nilotinib in inhibiting proliferation, viability, and clonogenic function of TKI-insensitive quiescent CD34+ CML chronic phase (CP) cells while normal CD34+ cells retained their clonogenic capacity in response to this combination therapy in vitro. Mechanistically, the pro-apoptotic activity of IB likely resides in part through its dual ability to block NF-κB and re-activate the tumor suppressor protein phosphatase 2A (PP2A). Combining BCR-ABL1 kinase inhibition with NADPH oxidase blockade may be beneficial in eradication of CML and worthy of further investigation.

Natural killer (NK) cells mediate immune responses against cancer; however, NK cell quantitative and functional defects are features of cancers including chronic myelogenous leukemia (CML). As increased numbers of activated NK cells were found in CML patients in treatment-free remission, the understanding of the molecular events inhibiting NK proliferation and function may lead to the development of NK cell-based therapies against drug-resistant cancer stem cells. Because altered miRNA expression and inactivation of the protein phosphatase 2A (PP2A) tumor suppressor are also features of cancer, and SET-dependent PP2A inhibition is essential for NK cell function, we hypothesized that increased expression of miR-300, a miRNA with antiproliferative activity, found inhibited in CML and targeting the PP2A inhibitor SET, accounts for impaired NK cell proliferation/activity. An initial analysis revealed that miR-300 levels were significantly higher in peripheral blood (PB) CD56+CD3- NK cells from CML patients at diagnosis compared to healthy individuals. As NK cell activity is regulated by the bone marrow microenvironment (BMM), we evaluated whether hypoxic conditions and/or cell-to-cell interaction influence NK cell proliferation and cytotoxic activity by modulating miR-300 intracellular levels. A marked and significant increase in miR-300 expression was detected in NK-92 and primary CD56+CD3- NK cells exposed to low O2 levels or cultured in the presence of conditioned medium (CM) or exosomes isolated from BM-derived primary mesenchymal stromal (MSC) and HS-5 MSC cells. As expected, increased miR-300 levels correlated with decreased SET levels and markedly reduced NK cell number. Interestingly, miR-300-induced growth inhibition was rescued in NK cells treated with CM/exosomes from HS-5 cells expressing an anti-miR-300 lentivirus. Notably, qRT-PCR indicated that miR-300 was contained in MSC exosomes. Functionally, exposure to MSCs (CM or exosomes) inhibited NK cell IL-12/IL-18-induced IFN-γ production and cytotoxic activity against K562 CML-BC cells in a miR-300-dependent manner. Accordingly, miR-300 lentiviruses and/or CpG-miR-300 oligonucleotides inhibited SET expression, reduced proliferation and suppressed spontaneous cytotoxicity of NK-92 and/or primary NK cells, likely through reactivation of PP2A. Because BM hypoxic conditions and MSCs significantly contribute to decreased NK cell number and cytotoxic activity through upregulation of miR-300, its genetic or pharmacologic inhibition may result in reactivation of NK cell activity against leukemic stem/progenitor cells. This work is supported in part by NIH-NCI RO1CA163800. Citation Format: Rossana Trotta, Giovannino Silvestri, Lorenzo Stramucci, Martin Guimond, Guido Marcucci, Xiaoxuan Fan, Maria R. Baer, Danilo Perrotti. Bone marrow microenvironment-induced miR-300 expression impairs natural killer cell proliferation and antitumor activity [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 4736.

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.

Tyrosine kinase inhibitors (TKIs) combined with chemotherapy significantly improved outcomes in adult Philadelphia-chromosome-positive (Ph+) B-cell Acute Lymphoblastic Leukemia (B-ALL). However, high relapse rates due to development of TKI resistance or chemotherapy-induced adverse effects remain the major therapeutic challenges. Furthermore, all TKIs are not effective against Ph+leukemia-initiating cells (LICs). The tumor suppressor protein phosphatase 2A (PP2A) is inactive in almost all solid and hematopoietic tumors. Suppression of PP2A activity correlates with poor outcome and disease progression, and largely relies on the aberrant expression of CIP2A, SET and/or SETBP1. SETBP1 was discovered as a SET-interacting protein, and recently described as mutated or overexpressed in several myeloid malignancies where it acts as an independent negative prognostic factor and as an inhibitor of PP2A, however its role in lymphoid malignancies is still unknown. Thus, we postulated that SETBP1 regulates survival and self-renewal of Ph+B-ALL LICs and progenitors through inhibition of PP2A. We reported that BCR-ABL1 kinase-dependent and -independent mechanisms induce SET-dependent PP2A inhibition in Ph+ (CMLand B-ALL) progenitors and quiescent TKI-resistant CML LICs, respectively, and that SET downregulation or pharmacologic (i.e. SET-interacting PP2A-activating drugs; PADs) restoration of PP2A activity strongly impaired malignant but not normal hematopoiesis by selectively killing Ph+progenitors (CML and ALL) and TKI-resistant quiescent stem (CML) cells. Here, we show that wild type SETBP1 is markedly induced in an imatinib (IM)-insensitive manner in primary CD34+CD19+ Ph+ B-ALL progenitors and Ph+ B-ALL cell lines (BV173 and SUP-B15) and barely detectable in CD34+ cells from CML patients in chronic and blastic phase. Overexpression of SETBP1 was found essential for PP2A inhibition in Ph+ B-ALL blasts. Accordingly, shRNA-dependent SETBP1 downregulation impaired clonogenic potential and self-renewal of CD34+CD19+ Ph+ B-ALL cells in CFC and serial replating assays. Furthermore, we have evidence that a SETBP1-SET/CIP2A inhibitory complex may exist in Ph+ cells, suggesting that SETBP1 might serve to recruit SET and CIP2A to suppress PP2A activity. Indeed, we found that CIP2A, like SET and SETBP1, is also overexpressed in CD34+CD19+ Ph+ B-ALL compared to CD34+CD19+cell from BM of healthy donors. Because, SETBP1 stabilizes SET and augments PP2A inhibition, and ectopic SETBP1 expression confers self-renewal to mouse myeloid progenitors and cooperates with BCR-ABL1 to induce a CML blast crisis-like disease in mice, our data suggest that aberrant SETBP1 expression might significantly contributes to the development of Ph+ B-ALL and persistence of TKI-resistant Ph+ B-ALL LICs. Disclosures Milojkovic: Ariad: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Roy:Paladin: Consultancy; Fate Therapeutics: Consultancy; Novartis: Consultancy; Kiadis Pharma: Research Funding.

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.

The persistence of tyrosine kinase inhibitor (TKI)-resistant malignant Philadelphia-positive (Ph+) hematopoietic stem cells (HSC) in chronic myelogenous leukemia (CML) TKI-treated patients in complete molecular remission, and the dismal prognosis of blast crisis CML indicate that the molecular mechanisms underlying its emergence, maintenance and progression are not totally dependent on the unrestrained kinase activity of BCR-ABL1 and might rely on other cell autonomous and/or microenvironmental signal capable of sustaining survival and self-renewal of the chronic phase and blast crisis Ph+ HSC compartment(s). We recently demonstrated that the Jak2/SET-PP2A/β-catenin pathway is essential for survival and self-renewal of quiescent TKI-resistant CD34+CD38- Ph+ HSC and that activation of such oncogenic signals requires the expression but not the activity of BCR-ABL1. Because microRNAs (miRNAs) are likely to control in a canonical and/or decoy manner the expression of different components of the Jak2 signalosome, this makes them an attractive target for further understanding the mechanisms of leukemogenesis and, perhaps, for developing new alternative therapies that selectively eradicate quiescent leukemic HSCs. In silico analysis revealed that a specific miR subset shares multiple targets of the BCR-ABL1/Jak2/SET-PP2A signalosome. Nanostring Array analysis performed on primary bone marrow cells from normal individuals and chronic phase or blast crisis CML patients revealed that expression of some of these miRNA is altered in CML. For example, expression of miR-300 and miR-101, which are predicted to simultaneously modulate directly Jak2, hnRNP-A1 and β-catenin and, indirectly, other molecules of the BCR-ABL1/Jak2/SET-PP2A/β-catenin pathway, is significantly inhibited in chronic phase CML and further decreases in advanced CML samples. Additionally, miR-300 expression is several folds lower in dividing (div. 1) compared to quiescent (CFSEMAX) CD34+ CML cells. Lentiviral-transduction of miR-300 in human BCR-ABL+ cell lines resulted in marked downmodulation of JAK2, β-Catenin hnRNPA1 and SET and, as expected, in increased PP2A activity. Moreover, ectopic miR-300 expression decreased reduced clonogenic potential and proliferation, and increased susceptibility to TKI (e.g. Imatinib) induced apoptosis. Interestingly, it appears that forced BCR-ABL1 expression in TF-1 leukemic cells decreases miR-300, consistent with the reported activation in these cells of the Jak2-SET-PP2A-β-catenin pathway. Altogether our results suggest that miR-300 expression and, potentially, that of other deregulated non-coding RNAs might be dispensable or deleterious for the phenotype of Ph+ progenitors and/or indispensable for that of Ph+ HSCs. Thus, experiments in BCR-ABL1 cell lines as well as primary stem and progenitor cell fractions are currently ongoing to assess the role of this and other miRNAs in survival, self-renewal/proliferation of CML stem and progenitor cells. Disclosures No relevant conflicts of interest to declare.

Background: CML is the most common myeloproliferative disease observed among adults, its 1st line of treatment is IM with a response rate ranging between 55 – 90%. In Qatar the resistance rate is higher than 45%. Our collaborators in Italy recently reported on the relation between CML and PTPRG. Methods: One cohort of patients (n=25, period=3years) receiving Imatinib was studied for haematological, cytogenetic molecular and biochemical abnormalities. Our collaborators in Italy examined different CML cell lines and an independent cohort of patients for the level of expression of PTPRG using QPCR, clonogenic assays, methylation-specific PCR, flow cytometry and western blotting. Results: Our team reported previously on the high rate of resistance of CML to IM (45%). During this forum the team is further reporting on the possible underlying mechanisms behind this resistance (see Al-Dewik et al at this forum) Despite a few positive findings, no pattern could be identified to delineate a significant underlying mechanism. Our collaborators in Italy, identified that down-regulation of PTPRG increased colony formation in the PTPRG+ve megakaryocytic MEG-01 and LAMA-84, but had no effect in the PTPRG-ve K562 and KYO-1. Its over-expression had an oncosuppressive effect in all four cell lines and is associated with inhibition of BCR/ABL-dependent signalling. PTPRG was down-regulated at the mRNA and protein levels in CML patients in both PB and BM, including CD34+ cells, and is re-expressed following molecular remission of disease. This re-expression was associated with loss of methylation of a CpG island of PTPRG promoter in 55% patients. In K562 cells, the hypomethylating agent 5-aza-2’-deoxycytidine induced PTPRG expression and caused inhibition of colony formation that was partially reverted by antisense-mediated down-regulation of PTPRG expression. Conclusions: Although this study was done on 2 independent patient populations, it suggests that in CML populations with high resistance rate it might be worth examining the PTPRG expression level and correlate it with the pattern of resistance. Our group has secured 3 years funding from QNRF (NPRP 4-157-3-052) to investigate PTPRG signalling in CML, including the study of a possible link among the high CML resistance and the PTPRG expression levels.