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Space travel has always been the man’s ultimate destination. With the ability of spaceflight though, came the realization that exposure to microgravity has lasting effects on the human body. To counteract these, many studies were and are undertaken, on multiple levels. Changes in cell growth, gene, and protein expression have been described in different models on Earth and in space. Extracellular vesicles, and in particular exosomes, are important cell-cell communicators, being secreted from almost all the cells and therefore, are a perfect target to further investigate the underlying reasons of the organism’s adaptations to microgravity. Here, we studied supernatants harvested from the CellBox-1 experiment, which featured human thyroid cancer cells flown to the International Space Station during the SpaceX CRS-3 cargo mission. The initial results show differences in the number of secreted exosomes, as well as in the distribution of subpopulations in regards to their surface protein expression. Notably, alteration of their population regarding the tetraspanin surface expression was observed. This is a promising step into a new area of microgravity research and will potentially lead to the discovery of new biomarkers and pathways of cellular cross-talk.

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 transcribed ultraconserved regions (T-UCRs) encode long non-coding RNAs implicated in human carcinogenesis. Their mechanisms of action and the factors regulating their expression in cancers are poorly understood. Here we show that high expression of uc.339 correlates with lower survival in 210 non-small cell lung cancer (NSCLC) patients. We provide evidence from cell lines and primary samples that TP53 directly regulates uc.339. We find that transcribed uc.339 is upregulated in archival NSCLC samples, functioning as a decoy RNA for miR-339-3p, -663b-3p, and -95-5p. As a result, Cyclin E2, a direct target of all these microRNAs is upregulated, promoting cancer growth and migration. Finally, we find that modulation of uc.339 affects microRNA expression. However, overexpression or downregulation of these microRNAs causes no significant variations in uc.339 levels, suggesting a type of interaction for uc.339 that we call “entrapping”. Our results support a key role for uc.339 in lung cancer., T-UCRs encode long non-coding RNAs implicated in human carcinogenesis, but the underlying mechanisms are poorly understood. Here, the authors identify uc.339 as an oncogene in lung cancer that is upregulated through the loss of TP53 and promotes Cyclin E activation by entrapping regulatory miRNAs.

The transcribed ultraconserved regions (T-UCRs) encode long non-coding RNAs implicated in human carcinogenesis. Their mechanisms of action and the factors regulating their expression in cancers are poorly understood. Here we show that high expression of uc.339 correlates with lower survival in 210 non-small cell lung cancer (NSCLC) patients. We provide evidence from cell lines and primary samples that TP53 directly regulates uc.339. We find that transcribed uc.339 is upregulated in archival NSCLC samples, functioning as a decoy RNA for miR-339-3p, -663b-3p, and -95-5p. As a result, Cyclin E2, a direct target of all these microRNAs is upregulated, promoting cancer growth and migration. Finally, we find that modulation of uc.339 affects microRNA expression. However, overexpression or downregulation of these microRNAs causes no significant variations in uc.339 levels, suggesting a type of interaction for uc.339 that we call “entrapping”. Our results support a key role for uc.339 in lung cancer.

Alzheimer's disease (AD) is a major public health challenge that faces an aging global population. Current drug treatment has demonstrated only symptomatic efficacy, leaving an unmet medical need for a new generation of disease-modifying therapies. Following the multitarget-directed ligand approach, a small library of coumarin-based derivatives was designed and synthesized as a follow-up to our studies on AP2238, aimed at expanding its biological profile. The coumarin substitution pattern at the 6- or 7-position was modified by introducing alkyl chains of variable lengths and with different terminal amino functional groups. 3-(4-{[Benzyl(ethyl)amino]methyl}phenyl)-6-({5-[(7-methoxy-6H-indeno[2,1-b]quinolin-11-yl)amino]pentyl}oxy)-2H-chromen-2-one, bearing the bulkiest amine, emerged as a non-neurotoxic dual acetylcholinesterase (AChE)/butyrylcholinesterase (BuChE) inhibitor, potentially suitable for the treatment of the middle stage of AD. Furthermore, the introduction of a diethylamino spacer, as in 3-(4-{[benzyl(ethyl)amino]methyl}phenyl)-6-{[5-(diethylamino)pentyl]oxy}-2H-chromen-2-one and 3-(4-{[benzyl(ethyl)amino]methyl}phenyl)-7-[4-(diethylamino)butoxy]-2H-chromen-2-one, led to nanomolar human AChE inhibitors endowed with significant inhibitory activity toward Aβ42 self-aggregation, whereas the reference compound was completely ineffective. Furthermore, 3-(4-{[benzyl(ethyl)amino]methyl}phenyl)-7-[4-(diethylamino)butoxy]-2H-chromen-2-one also showed promising neuroprotective behavior, which makes it a potential candidate for development into a disease-modifying agent.

The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase–independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression — but not activity — of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/β-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase–independent and PP2A-mediated inhibition of JAK2 and β-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1–positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/β-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs.

Summary Protein phosphatase 2A (PP2A), one of the main serine–threonine phosphatases in mammalian cells, maintains cell homoeostasis by counteracting most of the kinase-driven intracellular signalling pathways. Unrestrained activation of oncogenic kinases together with inhibition of tumour suppressors is often required for development of cancer. PP2A has been shown to be genetically altered or functionally inactivated in many solid cancers and leukaemias, and is therefore a tumour suppressor. For example, the phosphatase activity of PP2A is suppressed in chronic myeloid leukaemia and other malignancies characterised by aberrant activity of oncogenic kinases. Preclinical studies show that pharmacological restoration of PP2A tumour-suppressor activity by PP2A-activating drugs (eg, FTY720) effectively antagonises cancer development and progression. Here, we discuss PP2A as a druggable tumour suppressor in view of the possible introduction of PP2A-activating drugs into anticancer therapeutic protocols.

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.

Protein phosphatase-2A (PP2A) is one of the major cellular serine-threonine phosphatases and is involved in the regulation of cell homeostasis through the negative regulation of signaling pathways initiated by protein kinases. As several cancers are characterized by the aberrant activity of oncogenic kinases, it was not surprising that a phosphatase like PP2A has progressively been considered as a potential tumor suppressor. Indeed, multiple solid tumors (e.g. melanomas, colorectal carcinomas, lung and breast cancers) present with genetic and/or functional inactivation of different PP2A subunits and, therefore, loss of PP2A phosphatase activity towards certain substrates. Likewise, impaired PP2A phosphatase activity has been linked to B-cell chronic lymphocytic leukemia, Philadelphia-chromosome positive acute lymphoblastic leukemia and blast crisis chronic myelogenous leukemia. Remarkably, drugs such as forskolin, 1,9-dideoxy-forskolin and FTY720 which lead to PP2A activation effectively antagonize leukemogenesis in both in vitro and in vivo models of these cancers. Thus, PP2A is now in the spotlight as a highly promising drugable target for the development of a new series of anticancer agents potentially capable of overcoming drug-resistance induced in patients by continuous exposure to kinase inhibitor monotherapy. Herein, we review current knowledge of PP2A biology and function with particular emphasis on its tumor suppressor activity and possible therapeutic implications in cancer.

Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are aberrantly regulated at transcriptional or posttranslational levels by the constitutive kinase activity of the BCR/ABL oncoprotein. As a result, altered expression/function of RBPs leads to increased resistance to apoptotic stimuli, enhanced survival, growth advantage, and differentiation arrest of CD34+ progenitors from patients in CML blast crisis. Here, we identify the mRNAs bound to the hnRNP-A1, hnRNP-E2, hnRNP-K, and La/SSB RBPs in BCR/ABL-transformed myeloid cells. Interestingly, we found that the mRNA encoding the transcription factor E2F3 associates to hnRNP-A1 through a conserved binding site located in the E2F3 3′ untranslated region (UTR). E2F3 levels were upregulated in CML-BCCD34+ in a BCR/ABL kinase- and hnRNP-A1 shuttling-dependent manner. Moreover, by using shRNA-mediated E2F3 knock-down and BCR/ABL-transduced lineage-negative bone marrow cells from E2F3+/+ and E2F3-/- mice, we show that E2F3 expression is important for BCR/ABL clonogenic activity and in vivo leukemogenic potential. Thus, the complexity of the mRNA/RBP network, together with the discovery of E2F3 as an hnRNP-A1-regulated factor, outlines the relevant role played by RBPs in posttranscriptional regulation of CML development and progression. © 2008 by The American Society of Hematology.

In t(8;21) acute myeloid leukemia (AML), the AML1/ETO fusion protein promotes leukemogenesis by recruiting class I histone deacetylase (HDAC)-containing repressor complex to the promoter of AML1 target genes. Valproic acid (VPA), a commonly used antiseizure and mood stabilizer drug, has been shown to cause growth arrest and induce differentiation of malignant cells via HDAC inhibition. VPA causes selective proteasomal degradation of HDAC2 but not other class I HDACs (i.e., HDAC 1, 3, and 8). Therefore, we raised the question of whether this drug can effectively target the leukemogenic activity of the AML1/ETO fusion protein that also recruits HDAC1, a key regulator of normal and aberrant histone acetylation. We report here that VPA treatment disrupts the AML1/ETO-HDAC1 physical interaction, stimulates the global dissociation of AML1/ETO-HDAC1 complex from the promoter of AML1/ETO target genes, and induces relocation of both AML1/ETO and HDAC1 protein from nuclear to perinuclear region. Furthermore, we show that mechanistically these effects associate with a significant inhibition of HDAC activity, histone H3 and H4 hyperacetylation, and recruitment of RNA polymerase II, leading to transcriptional reactivation of target genes (i.e., IL-3) otherwise silenced by AML1/ETO fusion protein. Ultimately, these pharmacological effects resulted in significant antileukemic activity mediated by partial cell differentiation and caspase-dependent apoptosis. Taken together, these data support the notion that VPA might effectively target AML1/ETO-driven leukemogenesis through disruption of aberrant HDAC1 function and that VPA should be integrated in novel therapeutic approaches for AML1/ETO-positive AML.

Altered mRNA metabolism is a feature of many cancers including blast crisis chronic myelogenous leukemia. Indeed, loss of function of many tumor suppressors regulating cell proliferation, survival, and differentiation results from aberrant mRNA processing, nuclear export, and/or translation. Here, we summarize the effects of increased BCR/ABL oncogenic activity on the expression and function of RNA binding proteins (e.g., FUS, hnRNP A1, hnRNP E2, hnRNP K, and La/SSB) with posttranscriptional and translational regulatory activities and their importance for the phenotype of BCR/ABL-transformed hematopoietic progenitors. We also provide evidence that these studies not only advance our understanding on the molecular mechanisms contributing to tumor/leukemia emergence, maintenance, and/or progression but they also serve for the identification of novel molecular targets useful for the development of alternative therapies for imatinib-resistant and blast crisis chronic myelogenous leukemia and, perhaps, for other cancers characterized by similar alterations in the mRNA metabolism.

Monokines (i.e., interleukin [IL]-12, -18, and -15) induce natural killer (NK) cells to produce interferon-gamma (IFN-gamma), which is a critical factor for immune surveillance of cancer and monocyte clearance of infection. We show that SET, which is a potent inhibitor of protein phosphatase type 2A (PP2A) activity, is highly expressed in human CD56bright NK cells, which produce more IFN-gamma than CD56dim NK cells. SET was up-regulated upon monokine stimulation of primary human NK cells. Furthermore, ectopic overexpression of SET significantly enhanced IFN-gamma gene expression in monokine-stimulated NK cells. In contrast, RNAi-mediated suppression of SET expression renders NK cells inefficient in producing high levels of IFN-gamma in response to monokine costimulation. Mechanistically, suppression of PP2A activity by SET is important for IFN-gamma gene expression in NK cells. In fact, treatment of primary human NK cells with the PP2A activator 1,9-dideoxy-forskolin, as well as administration of the drug to C57BL/6 mice, significantly reduced NK-dependent IFN-gamma production in response to monokine treatment. Further, SET knockdown or pharmacologic activation of PP2A diminished extracellular signal-regulated kinase 1/2, p65RelA, signal transducer and activator of transduction 4 (STAT4), and STAT5 activity in monokine-stimulated NK cells, potentially contributing to the reduction in IFN-gamma gene expression. Thus, SET expression is essential for suppressing PP2A phosphatase activity that would otherwise limit NK cell antitumoral and/or antiinflammatory functions by impairing NK cell production of IFN-gamma.

High levels of microRNA-155 (miR-155) are associated with poor outcome in acute myeloid leukemia (AML). In AML, miR-155 is regulated by NF-κB, the activity of which is, in part, controlled by the NEDD8-dependent ubiquitin ligases. We demonstrate that MLN4924, an inhibitor of NEDD8-activating enzyme presently being evaluated in clinical trials, decreases binding of NF-κB to the miR-155 promoter and downregulates miR-155 in AML cells. This results in the upregulation of the miR-155 targets SHIP1, an inhibitor of the PI3K/Akt pathway, and PU.1, a transcription factor important for myeloid differentiation, leading to monocytic differentiation and apoptosis. Consistent with these results, overexpression of miR-155 diminishes MLN4924-induced antileukemic effects. In vivo, MLN4924 reduces miR-155 expression and prolongs the survival of mice engrafted with leukemic cells. Our study demonstrates the potential of miR-155 as a novel therapeutic target in AML via pharmacologic interference with NF-κB-dependent regulatory mechanisms. We show the targeting of this oncogenic microRNA with MLN4924, a compound presently being evaluated in clinical trials in AML. As high miR-155 levels have been consistently associated with aggressive clinical phenotypes, our work opens new avenues for microRNA-targeting therapeutic approaches to leukemia and cancer patients.

Background: How exosomic microRNAs (miRNAs) contribute to the development of drug resistance in the context of the tumor microenvironment has not been previously described in neuroblastoma (NBL). Methods: Coculture experiments were performed to assess exosomic transfer of miR-21 from NBL cells to human monocytes and miR-155 from human monocytes to NBL cells. Luciferase reporter assays were performed to assess miR155 targeting of TERF1 in NBL cells. Tumor growth was measured in NBL xenografts treated with Cisplatin and peritumoral exosomic miR-155 (n = 6 mice per group) CD163, miR-155, and TERF1 levels were assessed in 20 NBL primary tissues by Human Exon Arrays and quantitative real-time polymerase chain reaction. Student’s t test was used to evaluate the differences between treatment groups. All statistical tests were two-sided. Results: miR-21 mean fold change (f.c.) was 12.08 ± 0.30 (P < .001) in human monocytes treated with NBL derived exosomes for 48 hours, and miR-155 mean f.c. was 4.51 ± 0.25 (P < .001) in NBL cells cocultured with human monocytes for 48 hours. TERF1 mean luciferase activity in miR-155 transfected NBL cells normalized to scrambled was 0.36 ± 0.05 (P

MicroRNAs (miRNAs) regulate gene expression in cells and are frequently dys-regulated in human cancers. Recent evidence indicates that miRNAs are released by normal and cancer cells within microves...

Current treatments for acute myeloid leukemia (AML) are designed to target rapidly dividing blast populations with limited success in eradicating the functionally distinct leukemia stem cell (LSC) population, which is postulated to be responsible for disease resistance and relapse. We have previously reported high miR-126 expression levels to be associated with a LSC-gene expression profile. Therefore, we hypothesized that miR-126 contributes to “stemness” and is a viable target for eliminating the LSC in AML. Here we first validate the clinical relevance of miR-126 expression in AML by showing that higher expression of this microRNA (miR) is associated with worse outcome in a large cohort of older (≥60 years) cytogenetically normal AML patients treated with conventional chemotherapy. We then show that miR-126 overexpression characterizes AML LSC-enriched cell subpopulations and contributes to LSC long-term maintenance and self-renewal. Finally, we demonstrate the feasibility of therapeutic targeting of miR-126 in LSCs with novel targeting nanoparticles (NP) containing antagomiR-126 resulting in in vivo reduction of LSCs likely by depletion of the quiescent cell subpopulation. Our findings suggest that by targeting a single miR, i.e., miR-126, it is possible to interfere with LSC activity, thereby opening potentially novel therapeutic approaches to treat AML patients.

Altered mRNA translation has been recently associated with malignant transformation of several cell types, including hematopoietic cells. The deregulated kinase activity of the BCR/ABL oncoproteins, hallmark of chronic myelogenous leukemia (CML), enhances proliferation and survival, and arrests differentiation of hematopoietic progenitors by aberrantly modulating the activity of signaling molecules that control gene transcription, induce post-translational modifications and regulate translation of mRNAs into functional proteins. In this review, we discuss the mechanisms whereby BCR/ABL, by altering the expression/function of specific RNA-binding proteins and the activity of the translational machinery, impairs maturation and decreases susceptibility to apoptosis of myeloid progenitors.

The SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A), is overexpressed in leukemia. We evaluated the efficacy of SET antagonism in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cell lines, a murine leukemia model, and primary patient samples using OP449, a specific, cell-penetrating peptide that antagonizes SET's inhibition of PP2A.In vitro cytotoxicity and specificity of OP449 in CML and AML cell lines and primary samples were measured using proliferation, apoptosis, and clonogenic assays. Efficacy of target inhibition by OP449 was evaluated by immunoblotting and PP2A assay. In vivo antitumor efficacy of OP449 was measured in human HL-60 xenografted murine model.We observed that OP449 inhibited growth of CML cells including those from patients with blastic phase disease and patients harboring highly drug-resistant BCR-ABL1 mutations. Combined treatment with OP449 and ABL1 tyrosine kinase inhibitors was significantly more cytotoxic to K562 cells and primary CD34(+) CML cells. SET protein levels remained unchanged with OP449 treatment, but BCR-ABL1-mediated downstream signaling was significantly inhibited with the degradation of key signaling molecules such as BCR-ABL1, STAT5, and AKT. Similarly, AML cell lines and primary patient samples with various genetic lesions showed inhibition of cell growth after treatment with OP449 alone or in combination with respective kinase inhibitors. Finally, OP449 reduced the tumor burden of mice xenografted with human leukemia cells.We demonstrate a novel therapeutic paradigm of SET antagonism using OP449 in combination with tyrosine kinase inhibitors for the treatment of CML and AML.

The BCR-ABL1 oncoprotein is the cause of chronic myeloid leukemia and occurs as a consequence of the translocation t(9;22), a well-defined genetic event that results in the formation of the Philadelphia chromosome. While this genomic aberration is recognized to be the main culprit of the chronic phase of chronic myeloid leukemia, the natural clonal evolution of this myeloproliferative neoplasm involves the accumulation of secondary alterations through genomic instability. Thus, efforts to dissect the frequency and nature of the genomic events at diagnosis and at later stages are producing valuable insights into understanding the mechanisms of blastic transformation and development of resistance in chronic myeloid leukemia. The identification of alternative BCR-ABL1-dependent and BCR-ABL1-independent targets that sustain the survival of leukemic blasts and/or leukemia-initiating cells will facilitate the development of novel viable therapeutic options for patients who become resistant or intolerant to the currently available therapeutic options based on tyrosine kinase inhibitors.

Aberrant expression of the secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) gene, which encodes a matricellular protein that participates in normal tissue remodeling, is associated with a variety of diseases including cancer, but the contribution of SPARC to malignant growth remains controversial. We previously reported that SPARC was among the most upregulated genes in cytogenetically normal acute myeloid leukemia (CN-AML) patients with gene-expression profiles predictive of unfavorable outcome, such as mutations in isocitrate dehydrogenase 2 (IDH2-R172) and overexpression of the oncogenes brain and acute leukemia, cytoplasmic (BAALC) and v-ets erythroblastosis virus E26 oncogene homolog (ERG). In contrast, SPARC was downregulated in CN-AML patients harboring mutations in nucleophosmin (NPM1) that are associated with favorable prognosis. Based on these observations, we hypothesized that SPARC expression is clinically relevant in AML. Here, we found that SPARC overexpression is associated with adverse outcome in CN-AML patients and promotes aggressive leukemia growth in murine models of AML. In leukemia cells, SPARC expression was mediated by the SP1/NF-κB transactivation complex. Furthermore, secreted SPARC activated the integrin-linked kinase/AKT (ILK/AKT) pathway, likely via integrin interaction, and subsequent β-catenin signaling, which is involved in leukemia cell self-renewal. Pharmacologic inhibition of the SP1/NF-κB complex resulted in SPARC downregulation and leukemia growth inhibition. Together, our data indicate that evaluation of SPARC expression has prognosticative value and SPARC is a potential therapeutic target for AML.

The dismal outcome of blast crisis chronic myelogenous leukemia (CML-BC) patients underscores the need for a better understanding of the mechanisms responsible for the development of drug resistance. Altered expression of the anti-apoptoticBcl-xL has been correlated with BCR-ABL leukemogenesis; however, its involvement in the pathogenesis and evolution of CML has not been formally demonstrated yet. Thus, we generated an inducible mouse model in which simultaneous expression of p210-BCR-ABL1 and deletion of bcl-x occurs within hematopoietic stem and progenitor cells. Absence of Bcl-xL did not affect development of the chronic phase-like myeloproliferative disease, but none of the deficient mice progressed to an advanced phenotype, suggesting the importance of Bcl-xL in survival of progressing early progenitor cells. Indeed, pharmacological antagonism of Bcl-xL, with ABT-263, combined with PP242-induced activation of BAD markedly augmented apoptosis of CML-BC cell lines and primary CD34(+) progenitors but not those from healthy donors, regardless of drug resistance induced by bone marrow stromal cell-generated signals. Moreover, studies in which BAD or Bcl-xL expression was molecularly altered strongly support their involvement in ABT-263/PP242-induced apoptosis of CML-BC progenitors. Thus, suppression of the antiapoptotic potential of Bcl-xL together with BAD activation represents an effective pharmacological approach for patients undergoing blastic transformation.

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V617F) oncogene. PP2A inactivation occurs in a Jak2(V617F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V617F) inactivation/downregulation and impairs clonogenic potential of Jak2(V617F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V617F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V617F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V617F) also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V617F) MPNs.

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.

Oncogenic mutations of the receptor tyrosine kinase c-KIT play an important role in the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis, and some acute myeloid leukemias (AML). Although juxtamembrane mutations commonly detected in gastrointestinal stromal tumor are sensitive to tyrosine kinase inhibitors, the kinase domain mutations frequently encountered in systemic mastocytosis and AML confer resistance and are largely unresponsive to targeted inhibition by the existing agent imatinib. In this study, we show that myeloid cells expressing activated c-KIT mutants that are imatinib sensitive (V560G) or imatinib resistant (D816V) can inhibit the tumor suppressor activity of protein phosphatase 2A (PP2A). This effect was associated with the reduced expression of PP2A structural (A) and regulatory subunits (B55α, B56α, B56γ, and B56δ). Overexpression of PP2A-Aα in D816V c-KIT cells induced apoptosis and inhibited proliferation. In addition, pharmacologic activation of PP2A by FTY720 reduced proliferation, inhibited clonogenic potential, and induced apoptosis of mutant c-KIT+ cells, while having no effect on wild-type c-KIT cells or empty vector controls. FTY720 treatment caused the dephosphorylation of the D816V c-KIT receptor and its downstream signaling targets pAkt, pSTAT5, and pERK1/2. Additionally, in vivo administration of FTY720 delayed the growth of V560G and D816V c-KIT tumors, inhibited splenic and bone marrow infiltration, and prolonged survival. Our findings show that PP2A inhibition is essential for c-KIT–mediated tumorigenesis, and that reactivating PP2A may offer an attractive strategy to treat drug-resistant c-KIT+ cancers. Cancer Res; 70(13); 5438–47. ©2010 AACR.

We showed that Eμ-MiR-155 transgenic mice develop acute lymphoblastic leukemia/high-grade lymphoma. Most of these leukemias start at approximately 9 months irrespective of the mouse strain. They are preceded by a polyclonal pre–B-cell proliferation, have variable clinical presentation, are transplantable, and develop oligo/monoclonal expansion. In this study, we show that in these transgenic mice the B-cell precursors have the highest MiR-155 transgene expression and are at the origin of the leukemias. We determine that Src homology 2 domain–containing inositol-5-phosphatase (SHIP) and CCAAT enhancer-binding protein β (C/EBPβ), 2 important regulators of the interleukin-6 signaling pathway, are direct targets of MiR-155 and become gradually more down-regulated in the leukemic than in the preleukemic mice. We hypothesize that miR-155, by down-modulating Ship and C/EBPβ, initiates a chain of events that leads to the accumulation of large pre-B cells and acute lymphoblastic leukemia/high-grade lymphoma.

Udgivelsesdato: 2010-Mar-5 MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA's seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins.

Hypermethylation of 5′-cytosine-guanosine islands of tumor suppressor genes resulting in their silencing has been proposed to be a hallmark of various tumors. Modulation of DNA methylation with DNA methylation inhibitors has been shown to result in cancer cell differentiation or apoptosis and represents a novel strategy for chemotherapy. Currently, effective DNA methylation inhibitors are mainly limited to decitabine and 5-azacytidine, which still show unfavorable toxicity profiles in the clinical setting. Thus, discovery and development of novel hypomethylating agents, with a more favorable toxicity profile, is essential to broaden the spectrum of epigenetic therapy. Parthenolide, the principal bioactive sesquiterpene lactone of feverfew, has been shown to alkylate Cys38 of p65 to inhibit nuclear factor-κB activation and exhibit anti-tumor activity in human malignancies. In this article, we report that parthenolide 1) inhibits DNA methyltransferase 1 (DNMT1) with an IC50 of 3.5 μM, possibly through alkylation of the proximal thiolate of Cys1226 of the catalytic domain by its γ-methylene lactone, and 2) down-regulates DNMT1 expression possibly associated with its SubG1 cell-cycle arrest or the interruption of transcriptional factor Sp1 binding to the promoter of DNMT1. These dual functions of parthenolide result in the observed in vitro and in vivo global DNA hypomethylation. Furthermore, parthenolide has been shown to reactivate tumor suppressor HIN-1 gene in vitro possibly associated with its promoter hypomethylation. Hence, our study established parthenolide as an effective DNA methylation inhibitor, representing a novel prototype for DNMT1 inhibitor discovery and development from natural structural-diversified sesquiterpene lactones.

Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph1-positive) acute lymphocytic leukemia (ALL) are 2 fatal BCR/ABL-driven leukemias against which Abl kinase inhibitors fail to induce a long-term response. We recently reported that functional loss of protein phosphatase 2A (PP2A) activity is important for CML blastic transformation. We assessed the therapeutic potential of the PP2A activator FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), an immunomodulator in Phase III trials for patients with multiple sclerosis or undergoing organ transplantation, in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL(+) leukemias. Our data indicate that FTY720 induces apoptosis and impairs clonogenicity of imatinib/dasatinib-sensitive and -resistant p210/p190(BCR/ABL) myeloid and lymphoid cell lines and CML-BCCD34+ and Ph1 ALL(CD34+/CD19+) progenitors but not of normal CD34(+) and CD34(+)/CD19(+) bone marrow cells. Furthermore, pharmacologic doses of FTY720 remarkably suppress in vivo p210/p190(BCR/ABL)-driven [including p210/p190(BCR/ABL) (T315I)] leukemogenesis without exerting any toxicity. Altogether, these results highlight the therapeutic relevance of rescuing PP2A tumor suppressor activity in Ph1 leukemias and strongly support the introduction of the PP2A activator FTY720 in the treatment of CML-BC and Ph1 ALL patients.

Summary Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease (MPD) initiated by expression of the p210-BCR-ABL fusion protein. We demonstrate in a murine model of p210-BCR-ABL-induced MPD that gene targeting of Rac1 and Rac2 significantly delays or abrogates disease development. Attenuation of the disease phenotype is associated with severely diminished p210-BCR-ABL-induced downstream signaling in primary hematopoietic cells. We utilize NSC23766, a small molecule antagonist of Rac activation, to validate biochemically and functionally Rac as a molecular target in both a relevant animal model and in primary human CML cells in vitro and in a xenograft model in vivo, including in Imatinib-resistant p210-BCR-ABL disease. These data demonstrate that Rac is an additional therapeutic target in p210-BCR-ABL-mediated MPD.

The deregulated kinase activity of p210-BCR/ABL oncoproteins, hallmark of chronic myelogenous leukaemia (CML), induces and sustains the leukaemic phenotype, and contributes to disease progression. Imatinib mesylate, a BCR/ABL kinase inhibitor, is effective in most of chronic phase CML patients. However, a significant percentage of CML patients develop resistance to imatinib and/or still progresses to blast crisis, a disease stage that is often refractory to imatinib therapy. Furthermore, there is compelling evidence indicating that the CML leukaemia stem cell is also resistant to imatinib. Thus, there is still a need for new drugs that, if combined with imatinib, will decrease the rate of relapse, fully overcome imatinib resistance and prevent blastic transformation of CML. We recently reported that the activity of the tumour suppressor protein phosphatase 2A (PP2A) is markedly inhibited in blast crisis CML patient cells and that molecular or pharmacologic re-activation of PP2A phosphatase led to growth suppression, enhanced apoptosis, impaired clonogenic potential and decreased in vivo leukaemogenesis of imatinib-sensitive and -resistant (T315I included) CML-BC patient cells and/or BCR/ABL+ myeloid progenitor cell lines. Thus, the combination of PP2A phosphatase-activating and BCR/ABL kinase-inhibiting drugs may represent a powerful therapeutic strategy for blast crisis CML patients.

Altered mRNA translation is one of the effects exerted by the BCR/ABL oncoprotein in the blast crisis phase of chronic myelogenous leukemia (CML). Here, we report that in BCR/ABL+ cell lines and in patient-derived CML blast crisis mononuclear and CD34+ cells, p210BCR/ABL increases expression and activity of the transcriptional-inducer and translational-regulator heterogeneous nuclear ribonucleoprotein K (hnRNP K or HNRPK) in a dose- and kinase-dependent manner through the activation of the MAPKERK1/2 pathway. Furthermore, HNRPK down-regulation and interference with HNRPK translation-but not transcription-regulatory activity impairs cytokine-independent proliferation, clonogenic potential, and in vivo leukemogenic activity of BCR/ABL-expressing myeloid 32Dcl3 and/or primary CD34+ CML-BC patient cells. Mechanistically, we demonstrate that decreased internal ribosome entry site (IRES)-dependent Myc mRNA translation accounts for the phenotypic changes induced by inhibition of the BCR/ABL-ERK-dependent HNRPK translation-regulatory function. Accordingly, MYC protein but not mRNA levels are increased in the CD34+ fraction of patients with CML in accelerated and blastic phase but not in chronic phase CML patients and in the CD34+ fraction of marrow cells from healthy donors. Thus, BCR/ABL-dependent enhancement of HNRPK translation-regulation is important for BCR/ABL leukemogenesis and, perhaps, it might contribute to blast crisis transformation. (Blood. 2006;107:2507-2516)

Translational regulation by oncogenic proteins may be a rapid and efficient mechanism to modulate gene expression. We report here the identification of the CEBPB gene as a target of translational regulation in myeloid precursor cells transformed by the BCR/ABL oncogene. Expression of CEBPB was repressed in 32D-BCR/ABL cells and reinduced by imatinib (STI571) via a mechanism that appears to depend on expression of the CUG-repeat RNA-binding protein CUGBP1 and the integrity of the CUG-rich intercistronic region of c/ebpbeta mRNA. Constitutive expression or conditional activation of wild-type CEBPB induced differentiation and inhibited proliferation of 32D-BCR/ABL cells in vitro and in mice, but a DNA binding-deficient CEBPB mutant had no effect. The proliferation-inhibitory effect of CEBPB was, in part, mediated by the CEBPB-induced GADD45A gene. Because expression of CEBPB (and CEBPA) is low in the blast crisis (BC) stage of chronic myelogenous leukemia (CML) and is inversely correlated with BCR/ABL tyrosine kinase levels, these findings point to the therapeutic potential of restoring C/EBP activity in CML-BC and, perhaps, other types of acute leukemia.

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.

The dismal outcome of blast crisis chronic myelogenous leukemia (CML-BC) patients underscores the need for a better understanding of the mechanisms responsible for the development of drug-resistance and disease progression. We generated an inducible mouse model in which simultaneous expression of p210 BCR-ABL1 and deletion of bcl-x occurs within hematopoietic stem and progenitor cells. Absence of Bcl-xL did not affect the development of the chronic phase-like myeloproliferative disease; however, none of the deficient mice progressed to an advanced phenotype, suggesting the importance of the anti-apoptotic Bcl-xL signaling network for the progressing early progenitor cell survival. Indeed, pharmacologic inhibition of Bcl-xL with the Bcl-xL/Bcl-2 antagonist ABT-263, combined with activation of the Bcl-xL-regulator BAD, through the PP242-mediated inhibition of mTORC1/2, markedly augmented apoptosis of BCR-ABL1+ cell lines and primary CD34+ progenitors from CML-BC but not from healthy donors, regardless of the drug-resistance effect induced by bone marrow stromal cell-generated signals. Accordingly, shRNA-mediated downregulation of BAD and the BCR-ABL1-target hnRNP A1, which promotes translation of Bcl-xL but not Bcl-2, impaired the ability of PP242 to potentiate the effect of ABT-263, and mimicked the anti-leukemic activity of ABT-263, respectively, in primary leukemic CD34+ progenitors and/or CML-BC cell lines. Thus, suppression of the antiapoptotic potential of Bcl-xL together with activation of Bad represents a novel potential pharmacologic approach for treatment of those patients undergoing blastic transformation. Citation Format: Jason Harb, Paolo Neviani, Justin E. Ellis, Gregory J. Ferenchak, Brenda I. Chyla, Peter Hokland, Denis Claude Roy, Michael A. Caligiuri, Guido Marcucci, Claudia S. Huettner, Danilo Perrotti. Bcl-xL anti-apoptotic network is dispensable for emergence and maintenance of CML but required for disease progression, and represents an alternative target for halting survival of blast crisis CML progenitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3454. doi:10.1158/1538-7445.AM2013-3454

The unfolded protein response (UPR) is a mechanism by which normal cells react to endoplasmic reticulum (ER) stress to maintain cell homeostasis. ER stress is triggered by a variety of stimuli, including nutrient deprivation, oxidative stress and higher metabolic demand. This often results in the accumulation of unfolded or misfolded proteins in the ER lumen, a phenomenon that triggers the switch-on of the UPR. Thus, a complex network of pathways will act together to protect, adapt and recover the “injured” cells from ER stress.1 At molecular level, this translates into inhibition of protein translation and enhanced transcription of genes encoding molecular chaperones and other factors important for protein folding, degradation and quality control.1 If the damage to the ER persists over a prolonged period of time, apoptosis is normally evoked to eliminate damaged cells.2 Because cancer cells are generally exposed to a multitude of internal and external metabolic stressors, it is not surprising that molecular pathways regulating the cell response to ER stress have been found associated with autophagic and antiapoptotic signals and aberrantly activated in solid tumors and leukemias,1,3 two characteristics that make this pathway suitable to be used for therapeutic intervention. For example, a suitable target for anticancer drug development is represented by the ER chaperone GRP78; in fact, its high level of expression in a variety of tumors, including hepatocellular carcinoma, breast cancer and chronic myeloid leukemia (CML), is a strong indicator of a deregulated and, likely, constitutively active UPR.1,3-6 CML is characterized by the presence of the Philadelphia chromosome carrying the fusion oncogene BCR-ABL1.7 The presence of this constitutively active tyrosine kinase in myeloid progenitors is sufficient to induce and maintain their enhanced survival, a feature that is typical of the prolonged and indolent chronic phase (CP) of CML.7 While in the mid-’90s allogeneic stem cell transplantation was the only curative, albeit risky, option for CML, from early 2000, first- and second- and, soon, third-generation TKIs (i.e., imatinib, nilotinib, dasatinib, bosutinib and ponatinib) are the elective therapeutic choice for chronic phase patients, the majority of which achieve and maintain major or complete molecular response.7 However, in a small percentage of patients that are either refractory or become resistant to ABL1 tyrosine kinase inhibitors, CML undergoes blastic transformation, a still-fatal disease stage, historically termed blast crisis (BC) that is characterized by the increased expression and/or activity of BCR-ABL1 and the accumulation of secondary genetic and molecular abnormalities.7 Thus, it is therefore imperative to explore alternative routes that may be helpful to prevent the arising of resistance to TKIs and, most importantly, offer patients in CML-BC new-targeted therapeutic options that may either eliminate the leukemic cell clone or make it responsive to TKIs and other available drugs. In a recent issue of Cell Cycle, Kusio-Kobialka et al.8 describe for the first time that in CML there is a correlation between ER stress, CML progression and response to imatinib treatment. In particular, they found that in human CML cell lines and primary cells, the PKR-like ER-resident kinase (PERK) is activated in a BCR-ABL1 expression-dependent manner.8 PERK is one of the main initiators of the UPR and PERK-dependent phosphorylation of eIF2α impairs global cap-dependent mRNA translation, with the exception of ATF4 mRNA, whose product activates pathways controlling adaptation to stress and apoptosis.1 Importantly, the activation of the PERK-eIF2α pathway seems to follow the natural progression of the disease and is enhanced in cells derived from patients in CML-BC as opposed to patients in the chronic phase or to cells derived from healthy individuals.8 When BCR-ABL1-expressing cells were treated with imatinib, the authors saw a downregulation of PERK and eIF2α expression and phosphorylation levels in a dose-dependent manner, suggesting that the induction of the response to the ER stress may be mediated by BCR-ABL1 activity.8 By using dominant-negative mutants of PERK or eIF2α, the authors have also been able to show that the PERK-eIF2α pathway serves a pro-survival role in CML; in fact, cells expressing their dominant-negative forms show a decreased ability to form colonies in clonogenic assays and also seem to be more sensitive to imatinib-mediated cell death.8 In conclusion, this manuscript highlights the importance of exploring alternative pathways, like those involved in the UPR, as they might constitute the answer to overcoming the current therapeutic limitations we are facing in treating CML-BC and other acute leukemia patients.

Abstract 35 As tyrosine kinase inhibitors (TKIs) do not induce long-term response in blast crisis chronic myeloid leukemia (CML-BC) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), and are unable to kill quiescent Ph+ hematopoietic stem cells, alternative therapies targeting dysregulated pathways in BCR-ABL1+ acute leukemias are needed. CRM1, a karyopherin aberrantly overexpressed in several cancers, controls the nuclear export of proteins (e.g. ABL1, SET, p53, p21, FOXO and RB) that regulate normal and malignant hematopoietic cell survival, self-renewal and proliferation. Here we show that enhanced CRM1 expression also occurs in Ph+ leukemias and the clinical stage small molecule inhibitor of CRM1, KPT-330, has a detrimental effect on malignant but not normal hematopoietic progenitors. Specifically, a substantial upregulation (≥ 65% increase) in the expression levels of CRM1 was detected by immunoblot in CD34+ progenitors from bone marrow (BM) of leukemic primary samples (n=3), when compared to CD34+ progenitors from healthy (n=3) donors. Interestingly, CRM1 protein levels in CML CD34+ cells were markedly but not totally reduced by treatment with the ABL1 kinase inhibitor Imatinib (1μM, 72h), suggesting that BCR-ABL1-driven pathways are not the only factor contributing to CRM1 upregulation. Additionally, treatment of primary CML cells with KPT-330 (1μM, 72h) resulted in a 75% reduction in BCR-ABL1 kinase activity, consistent with an interrelationship between BCR-ABL1 and CRM1 activities. Finally, the ability of BCR-ABL1 to induce CRM1 expression was demonstrated upon ectopic BCR-ABL1 expression in myeloid 32Dcl3 precursor cells in which CRM1 protein levels became ∼10-fold higher. To determine the therapeutic relevance of CRM1 inhibition, cell survival was assessed in MACS-purified CD34+ progenitors. Treatment with KPT-330 resulted in ≥80% induction of apoptosis in Ph+ (n=5) CML (2μM, 72hr) and B-ALL (500nM, 72h) CD34+ cells. Conversely, KPT-330 exerted only a minimal effect on the survival of CD34+ progenitors from healthy donor BM (15–30% Annexin V positive, KPT-330 0.5–2μM, 72h). Consistent with the existence of BCR-ABL1-independent signaling leading to increased CRM1 expression, marked apoptosis was also observed in KPT-330-treated CD34+ progenitors isolated from a Ph-negative B-ALL patient. Thus, CRM1 inhibitor-based therapies might also benefit BCR-ABL1-negative leukemias. To formally determine the effects of KPT-330 on leukemic cell survival, methylcellulose-based clonogenic assays were performed. KPT-330 treatment (1μM) resulted in almost total suppression (97% reduction) of the clonogenic potential of leukemic but not normal (30% reduction) CD34+ progenitors. Because of evidence that CRM1 directly interacts with the protein phosphatase 2A (PP2A) inhibitor SET, and that CRM1 inhibition alters trafficking of hnRNP A1, a direct regulator of the SET-PP2A interplay in Ph+ leukemias, it is conceivable that the anti-leukemic activity of KPT-330 is, at least in part, mediated by PP2A activation. Indeed, KPT-330 treatment (1μM, 48hr) of BCR-ABL1+ cell lines resulted in full restoration of PP2A activity, comparable to levels observed in BCR-ABL1-negative cells. Accordingly, KPT-330 treatment (1μM, 12h) of 32Dcl3BCR-ABL1 cells caused a nuclear accumulation (4-fold increase) and cytoplasmic decrease (95% lower) of SET protein levels, as indicated by both confocal microscopy and immunoblots of subcellular fractions. Moreover, KPT-330-treated cells showed altered hnRNP A1 cellular distribution and overall downregulation. However, unexpectedly, hnRNP A1 protein levels were proportionally higher in the cytoplasm and lower in the nucleus, suggesting that the deleterious effect of CRM1 inhibition on hnRNP A1 might not depend on direct inhibition of hnRNP-A1 nuclear export. Because KPT-330 displays favorable ADME properties and has been shown to alleviate leukemic burden using in vivo models of different cancers, we are currently testing the anti-leukemic activity of this compound in a mouse model of CML-BC. We do expect a profound inhibition of leukemogenesis, and all treated mice are currently alive with no signs of toxicity. Thus, CRM1 inhibition by KPT-330 represents a potential new therapeutic avenue which could easily be exploited in malignancies like CML-BC and Ph+ ALL that show a dismal outcome to current available therapies. Disclosures: Walker: Karyopharm Therapeutics Inc: Research Funding. Landesman:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Perrotti:Karyopharm Therapeutics Inc: Research Funding.

Abstract 773 Altered expression of SPARC, [secreted protein, acidic, cysteine-rich (osteonectin)] a gene encoding a protein that modulates cell-matrix interactions, has been reported in cancer and leukemia, but its precise contribution to malignant transformation is unknown. We previously reported that SPARC was among the most upregulated genes in the gene-expression profile (GEP) associated with IDH2-R172 mutations (mut; Marcucci et al. JCO 2010;28:2348) and among the most downregulated genes associated with NPM1 mut (Becker et al. JCO 2010;28:596) in cytogenetically normal (CN) AML. Both mutations have strong but opposite impact (IDH2-R172 mut, adverse; NPM1 mut, favorable) on clinical outcome. Thus, we hypothesized that SPARC may act as an oncogene and contribute to AML aggressiveness. To determine functional and mechanistic activities of SPARC in AML, we conducted gain- and loss-of-function experiments in cell lines and primary blasts collected from patients under the OSU Leukemia Tissue Bank protocol. In vitro, ectopic expression and knockdown of SPARC in THP-1 and Kasumi-1 AML cells led to increase (2.5-fold P=0.03) and decrease (45%, P=0.03) in colony forming ability, respectively. Clonogenic assays in SPARC- vs empty vector (EV)-infected AML blasts also showed 2-fold increase in colony-formation (P=.01) supporting a role of SPARC overexpression in promoting leukemia growth. The leukemogenic activity of the SPARC protein was likely mediated by its binding to αvβ3 integrin and in turn by the activation of the kinase ILK. This induced GSK3β phosphorylation (p) and in turn activation of β-catenin, previously reported to contribute to AML. Indeed, while ILK knockdown abolished SPARC-induced p-GSK3β, ectopic SPARC expression led to a 4-fold increased p-GSK3β, measured by p-GSK3β (Ser-9) immunoblot assay, and to stabilization of β-catenin, measured by p-β-catenin(Ser-552) and p-β-catenin(Ser-675) immunoblot assays in AML blasts. As a result, we observed increase in translocation of β-catenin to the nucleus in SPARC overexpressing cells by confocal microscopy. Nuclear β-catenin led to increased activity of the TCF/LEF transcription factors as shown by a TCF/LEF luciferase reporter assay (5.8-fold, P The results in the preclinical models prompted us to evaluate whether higher SPARC expression associated with aggressive human disease. Thus, SPARC expression levels were measured by NanoString nCounter assay in a cohort of CN-AML patients (pts; n=362; age, 18–83 years) comprehensively characterized for mutations in the NPM1, FLT3, CEBPA, WT1, TET2, MLL, IDH1, IDH2, RUNX1, ASXL1 and DNMT3A genes, and treated with cytarabine-anthracycline based regimens. Higher SPARC expressers were more likely classified in the European Leukemia Net (ELN) Intermediate-I (CEBPA wt, NPM1 mut with FLT3-ITD, or NPM1 wt) than the ELN Favorable Genetic Group (CEBPA mut and/or NPM1 mut without FLT3-ITD; P60 years); shorter DFS (P=.07), once adjusted for FLT3-ITD, RUNX1, WBC and age group; and shorter OS (P In conclusion, high SPARC expression contributes to aggressive AML growth via β-catenin activation-mediated mechanisms. SPARC overexpression independently predicted worse outcome in CN-AML pts, and may represent a potentially novel prognostic marker and therapeutic target in AML. Disclosures: No relevant conflicts of interest to declare.

Tyrosine kinase inhibitor (TKI)-based therapies do not induce long-term response in myeloid or lymphoid blast crisis (BC) chronic myelogenous leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). Increased expression/activity of nucleocytoplasmic-shuttling heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, E2 and K, are critical for the expression of factors (e.g. SET/PP2A, C/EBPβ/miR-328, and c-Myc) regulating proliferation and survival of Ph+ progenitors. Because the karyopherin CRM1 controls nuclear export of hnRNPs, we assessed the therapeutic potential of CRM1 inhibitors in CML-BC and Ph+ ALL models. Thus, myeloid 32D-p210BCR-ABL1 and lymphoid Baf3-p190BCR-ABL1 progenitors were exposed to the CRM1 selective & potent inhibitors of nuclear export (SINE) KPT-185 and KPT-207. MTT viability assays revealed that KPT-185 and KPT-207 decreased cell viability by ∼80% at concentrations ranging from 150-350 nM. The KPT-SINE not only induced killing, but also affected cytokine-independent growth of BCR-ABL1+ cells: proliferation was inhibited 89% and 81% by KPT-185 and KPT-207, respectively. Notably, growth and survival of non-transformed 32Dcl3 and BaF3 cells was not affected (70-100% viable cells) by KPT-SINE. As expected, treatment (1 μM; 48h) with these inhibitors altered the nuclear/cytoplasmic ratio of hnRNPs important for BCR-ABL1 leukemogenesis. As a result, treatment of BCR-ABL1+ cells with KPT-185 and KPT-207 (1 μM; 48h) resulted in 75% and 50% suppression of BCR-ABL1 expression and kinase activity, respectively. Furthermore, KPT-207 also reduced Myc expression in 32D-p210BCR-ABL1 cells; this is consistent with the potential interference of KPT-207 with the proliferation/survival signals triggered by the BCR-ABL1/MAPK/hnRNP K/Myc pathway in CML-BC progenitors. Because both KPT-185 and KPT-207 significantly alter hnRNP A1 localization, which is important for regulation of the PP2A inhibitor SET and, therefore, for BCR-ABL1 leukemogenesis, we assessed whether KPT-207 and KPT-185 negatively regulate PP2A activity. Indeed, treatment with KPT-207 and KPT-185 (250 nM; 48h) restored PP2A activity in 32D-p210BCR-ABL1 cells to levels similar to those detected in non-transformed 32Dcl3 cells. Although further investigation of KPT-207 and KPT-185 mechanism of action and assessment of their biologic/therapeutic effects in CML-BC and Ph+ ALL mouse models and primary leukemic and normal progenitors is currently ongoing, it is safe to conclude that selective nuclear export (SINE CRM1) inhibitors represent potentially powerful therapeutic tools that, if used alone or in combination with TKIs, might lead to sustained complete molecular remission in CML-BC and Ph+ ALL patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3839. doi:1538-7445.AM2012-3839

Abstract 3758 Despite the remarkable response in chronic phase CML, tyrosine kinase inhibitor (TKI)-based therapies do not induce long-term response in myeloid or lymphoid blast crisis CML (CML-BC) and Ph+ ALL, and are unable to kill quiescent Ph+ hematopoietic stem cells. We reported that CML disease progression is characterized by a BCR-ABL1 dose- and kinase-dependent increase in the expression/activity of nucleocytoplasmic-shuttling of heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, E2 and K, which are essential post-transcriptional and translational modulators of critical regulators of cell proliferation, survival and differentiation of CD34+ CML-BC and/or CD34+/CD19+ Ph+ ALL progenitors including SET/PP2A, E2F3, Bcl-xL, C/EBPa, miR-328, and c-Myc. The karyopherin (a class of proteins involved in regulating nuclear import/export) chromosome region maintenance 1 (CRM1) controls the nuclear export of several hnRNPs, and because growth/survival of CML-BC and Ph+ ALL progenitors requires the aberrant cytoplasmic activity of hnRNPs A1, E2 and/or K, we have explored the therapeutic potential of CRM1 inhibitors in p210 and p190 BCR-ABL1+ cell line models of CML-BC and Ph+ ALL, respectively. Thus, the cytokine-dependent myeloid 32Dcl3 and lymphoid BaF3, and the derived cytokine-independent 32D-p210BCR-ABL1 and Baf3-p190BCR-ABL1 mouse progenitors were exposed for 48 hr to different concentrations (0–10 μM) of the CRM1 selective & potent inhibitors of nuclear export (SINE) KPT-185 and KPT-207. MTT viability assays (n=3) revealed that KPT-185 and KPT-207 decreased viability ∼80% in 32D-p210BCR-ABL1 cells (KPT-185≥207) at concentrations ranging from 150–350 nM. Similar results were obtained in Baf3-p190BCR-ABL1 cells as the EC50 was 300nM for KPT-185 and KPT-207. The KPT-SINE not only induced killing, but also affected cytokine-independent growth of BCR-ABL1+ cells: proliferation was inhibited 89% and 81% by KPT-185 and KPT-207, respectively. Notably, growth and survival of non-transformed 32Dcl3 and BaF3 cells was not affected (70–100% viable cells) at concentrations (150–350 nM) of KPT-185 and KPT-207 that impair survival of BCR-ABL1+ cells. Mechanistically, we found that KPT-SINE CRM1 inhibitors altered the nuclear/cytoplasmic ratio of hnRNPs important for BCR-ABL1 leukemogenesis. Indeed, in KPT-207-treated (1 μM; 48h) BCR-ABL1+ cells hnRNP A1, E2 and K accumulated in the cytoplasm. Likewise, KPT-185 treatment (1 μM; 48h) resulted in hnRNP A1 being sequestered in the nucleus whereas hnRNP E2 distribution was not altered and, unexpectedly, levels of hnRNP K expression were markedly decreased in both subcellular compartments. Interestingly, treatment of BCR-ABL1+ cells with KPT-185 and KPT-207 (1 μM; 48h) resulted in 75% and 50% suppression of BCR-ABL1 expression and kinase activity, respectively. Furthermore, KPT-207 also reduced Myc expression in 32D-p210BCR-ABL1 cells; this is consistent with the potential interference of KPT-207 with the proliferation/survival signals triggered by the BCR-ABL1/MAPK/hnRNP K/Myc pathway in CML-BC progenitors. Because both KPT-185 and KPT-207 significantly alter hnRNP A1 localization in addition to impairing BCR-ABL1 expression/activity, and suppression of PP2A tumor suppressor activity is essential for both p210 and p190 BCR-ABL1-driven leukemogenesis, it is highly plausible that KPT-207 and KPT-185 negatively regulate the BCR-ABL1-dependent and hnRNP A1-mediated induction of the PP2A inhibitor SET thereby rescuing PP2A phosphatase activity, which in turn decreases BCR-ABL1 expression and kinase activity, and triggers in vitro and in vivo apoptosis of primary CML-BC and Ph+ ALL progenitors. Indeed, treatment with KPT-207 and KPT-185 (250 nM; 48h) restored PP2A activity in 32D-p210BCR-ABL1 cells to levels similar to those detected in non-transformed 32Dcl3 cells. Although further investigation of KPT-207 and KPT-185 mechanism of action and assessment of their biologic/therapeutic effects in CML-BC and Ph+ ALL mouse models and primary leukemic and normal progenitors is currently ongoing, it is safe to conclude that selective nuclear export (SINE CRM1) inhibitors represent potentially powerful therapeutic tools that, if used alone or in combination with TKIs, might lead to sustained complete molecular remission in CML-BC and Ph+ ALL patients. Disclosures: Shacham: Karyopharm: Equity Ownership. Kauffman:Karyopharm: Equity Ownership.

Abstract 2494 In CD34+ bone marrow (BM) progenitors from Polycythemia Vera (PV) patients and Jak2V617F-expressing cell lines, loss of protein phosphatase 2A (PP2A) tumor suppressor activity is essential for their survival and clonogenic potential, and it depends on signals generated by the mutated Jak2 oncogenic kinase that, in turn, is also negatively regulated by PP2A. Additionally, we showed (Oaks J.J. et al., ASH 2010) that suppression of PP2A requires expression of the Jak2-regulated PP2A inhibitor SET, and it can be reverted by clinically-relevant Jak2 inhibitors or by the sphingosine analog FTY720, which reactivates PP2A and inhibits Jak2V617F expression/activity upon binding/sequestration of SET. Here we report that, unlike the effect of BCR-ABL1 in CML, SET levels do not increase in normal CD34+ BM progenitors engineered to express Jak2V617F; however, SET knock-down restores PP2A activity in both Jak2V617F cell lines and CD34+ PV progenitors, suggesting a post-translational control of SET activity. By using Jak2 and PI-3K kinase inhibitors and SET mutants, we found that PP2A inactivation in PV depends on the Jak2V617F- and PI-3Kγ-induced SET phosphorylation on serine 24. Indeed, a ∼93% restoration of PP2A activity and suppression of SET phosphorylation is achieved upon exposure of Jak2V617F+ cells to the specific PI-3Kγ (AS-604850, 1μM) inhibitor. Likewise, ∼80% loss of SET inhibitory activity is observed upon transduction of S24A but not S9A SET mutant that, albeit essential for PP2A inhibition in other cell types, behaved as wild type SET. Because both Jak2 and PI-3Kγ may regulate nitric oxide synthase (NOS2) that, in turn, inhibits PP2A activity upon induction of peroxynitrate (PN)-mediated nitration of PP2A tyrosine residues with negative regulatory function, we assessed the effect of NOS/PN modulation on the Jak2V617F-dependent inactivation of PP2A. Treatment of Jak2V617F- expressing BaF3 cells with NO donors (1 mM DPTA NONOate and 10μM SIN-1) or PN (500 μM) resulted in 2.7- and 28-fold induction of PP2A activity, respectively. Accordingly, 80% inhibition of PP2A activity was observed in parental BaF3 cells upon inhibition of NOS2 by 3-bromo-7-nitroindazole (5μM). Thus, Jak2V617F inhibits PP2A by inducing SET phosphorylation and inhibiting PP2A tyrosine nitration. Notably, co-existence of both mechanisms of Jak2V617F-dependent PP2A inactivation is also supported by the evidence that NOS2 levels increase upon shRNA-mediated downregulation of SET expression, and by the marked decrease in SET phosphorylation and increase in PP2A tyrosine nitration detected in Jak2V617F cells treated with FTY720 (2.5 mM). Interestingly, similar effects were observed upon exposure of Jak2V617F cells to non-phosphorylatable and non-immunosuppressive FTY720 derivatives (e.g. OSU-2S, QC-FTY720 and SR-FTY720) but not in cells treated with the immunosuppressive phosphorylated FTY720 (FTY720-P). In agreement with our findings indicating that the anti-leukemic activity of FTY720 in Jak2V617F+ primary progenitors and cell lines does not require the sphingosine kinase 2-dependent conversion of FTY720 into FTY720-P and, consequently, its interaction with the S1PR1 receptor (Oaks JJ. et al., ASH 2010), we have now evidence showing that a synthetic FTY720-P, unlike FTY720, impairs (81% inhibition) PP2A activity in normal hematopoietic progenitors, and that the major part of the intracellular FTY720 remains non-phosphorylated. By using S1PR1-selective agonists and S1PR1 receptor knock-down, we also found that inhibition of PP2A activity by synthetic FTY720-P is mediated by the S1PR1-dependent triggering and activation of Jak2 and PI-3Kγ. Accordingly, co-treatment of normal hematopoietic progenitor cells with FTY720-P and either the Jak2 inhibitor AG490 or the PI-3Kγ inhibitor AS-604850 antagonized the inhibitory effects of FTY720-P and restored PP2A activity to levels similar to those detected in untreated cells. Altogether our data indicate that the anti-leukemic effects of FTY720 in CD34+ PV progenitors depends on its ability to antagonize the Jak2V617F- and S1PR1-induced and PI-3Kγ-mediated inactivation of PP2A through inhibition of the PI-3Kγ-dependent SET serine 24 phosphorylation, and via binding/sequestration of phosphorylated SET that, in turn, leads to enhancement of NOS/PN-mediated PP2A activation by tyrosine nitration. Disclosures: No relevant conflicts of interest to declare.

Abstract 2738 Tyrosine kinase inhibitors (TKIs) have become frontline therapy for CML; however, alternative therapies are required, as TKIs do not induce long-term response in CML patients undergoing blastic transformation and are ineffective against Philadelphia-positive (Ph+) quiescent stem cells, which show innate resistance to BCR-ABL1 kinase inhibitors. Therapeutic targets of interest are survival factors conferring resistance to TKI-induced apoptosis and/or those increasing proliferation of leukemic progenitors. We previously reported (Harb JG et al., ASH 2007) that genetic inactivation of Bcl-x did not inhibit BCR-ABL1 leukemogenesis in an inducible mouse model of CML. Thus, we hypothesize that BCR/ABL mediated post-translational modification and inactivation of pro-apoptotic BAD negates the requirement for the anti-apoptotic function of Bcl-xL in stem/progenitor cells from SCLtTA-BCR/ABL1/Bcl-x−/− mice. Following this rationale, we tested if simultaneous pharmacologic BAD activation and Bcl-xL inhibition may be an efficient way of killing CML stem/progenitor cells. To test this, loss of Bcl-xL function with increased levels of active BAD was achieved by expressing Bcl-x shRNA in 32D-BCR/ABL mouse myeloid precursors that were then treated with LY294002 (LY), which suppresses the inhibitory effects of PI-3K/Akt activation on BAD. Flow cytometric analysis of Annexin V+ cells revealed that levels of apoptosis were three times higher in BCR-ABL1+ cells expressing the Bcl-x shRNA when compared with vector-transduced BCR-ABL1+ cells. As expected, increased levels of dephosphorylated (active) BAD at the mitochondrial membrane were found in LY-treated BCR-ABL+ cells. Interestingly, co-treatment of Bcl-x shRNA-expressing BCR-ABL1+ cells with LY and the Bcl-xL/Bcl-2 antagonist ABT-263 (ABT) did not further promote apoptosis, suggesting that decreased survival of BCR-ABL1+ cells was dependent on downregulation of Bcl-xL and not Bcl-2. To determine efficacy of combined pharmacologic Bcl-xL inhibition and BAD activation, 32D-BCR/ABL and K562 cells were treated with compounds expected to activate BAD upon inhibition of PI-3K/Akt/mTOR-generated signals, used alone or in combination with ABT. Individually, at suboptimal doses, LY, Rapamycin (RAP), mTORC1/2 inhibitor PP242, and ABT were tolerated with apoptosis levels lower than 20%. Notably, when combined with ABT, all three efficiently induced apoptosis (∼90% Annexin V+) of BCR-ABL1+ cells. As with LY, increased levels of active BAD were found at the mitochondrial membrane of RAP- and PP242-treated BCR-ABL1+ cells. We found that PP242 downregulated p-Akt (92%), Mcl-1 (67%) and Bcl-xL (51%) more efficiently than RAP or LY. It has been shown that PP242 impairs the clonogenic potential of TKI-resistant mononuclear BM CML-BC cells; however, its effects when used alone or in combination with ABT on survival of normal and leukemic hematopoietic stem (HSCs) and progenitor cells is still unknown. Thus, HSC-enriched (CD34+/CD38-) and progenitor (CD34+) CML-BC cell fractions were isolated from bone marrow and peripheral blood and used in colony forming (CFC) assays with ABT, PP242 or ABT/PP242. ABT alone did not suppress colony formation of Ph+ CD34+/CD38− cells, while PP242 reduced it by nearly 50%. Conversely, ABT/PP242 combination decreased Ph+ stem and progenitor colony formation by ∼80%. Furthermore, the self-renewal of Ph+ CD34+/CD38− cells was markedly impaired by ABT/PP242 as demonstrated by the 80% decrease in replating efficiency. To assess if non-leukemic stem cells would tolerate ABT/PP242, colony assays were performed with LSK from wild type mice treated with ABT, PP242, RAP and ABT/PP242. We did not find a significant effect of ABT or PP242 on clonogenic potential when given as single agents. More importantly, combined treatment decreased CFC output by only 35% while RAP, which has an acceptable toxicity profile as it has been used in clinical trials for patients unresponsive to TKIs, decreased LSK colony forming potential by 50%. In summary, our data showing that combined treatment with the mTORC1/2 inhibitor/BAD activator PP242 and the BCl-xL/Bcl-2 antagonist ABT-263 markedly induces apoptosis of BCR-ABL+ cell lines, in HSCs and in progenitors from CML-BC patients. This approach warrants further pre-clinical investigation aimed at inclusion in clinical protocols for treating blast crisis CML. Disclosures: No relevant conflicts of interest to declare.

Abstract 775 We have shown (Oaks JJ et al. ASH 2009) that the tumor suppressor Protein Phosphatase 2A (PP2A) is functionally inactivated by Jak2V617F in cell line models of Jak2V617F myeloproliferative disorders (MPD) and Jak2V617F-transduced primary mouse bone marrow cells. Inhibition of Jak2 (600 nM Jak Inhibitor I; 50 μM AG490; 10h) or treatment with the PP2A activator FTY720 (2.5μM, 24 hours) restored PP2A activity that caused loss of Jak2V617F protein/activity, impaired Jak2V617F-driven colony formation, and induced apoptosis of Jak2V617F+ but not normal myeloid cells. Notably, FTY720 is a sphingosine analog suggested by the FDA to treat patients with Multiple Sclerosis due to its immunosuppressive activity when phosphorylated by sphingosine kinase 2 (SPHK2). Here we show that FTY720 treatment of CD34+ primary bone marrow cells from JakV617F+ PV patients (n=3) also rescued PP2A activity, induced Jak2 downregulation and significantly impaired cytokine-dependent clonogenic potential. Thus, FTY720 could be used as an alternative to Jak2 inhibitors, as in vitro and in animal assays showed that FTY720 (2.5μM) is not toxic against normal human myeloid progenitors while decreasing survival of CD34+ progenitors from MPD patients. To find out whether FTY720 uses the same mechanism to exert its immunosuppressive and anti-leukemic activities, we determined if the conversion of FTY720 into its phosphorylated form is important for rescuing PP2A activity in Jak2V617F-expressing cells. Impaired FTY720-P conversion by exposure to the SPHK inhibitor dimethylsphingosine (2.5μM, 6 hours) did not affect the ability of FTY720 to activate PP2A. Also, a synthetically phosphorylated FTY720 (FTY720-P, 2.5μM, 6 hours) was unable to activate PP2A or exert any anti-leukemic activity, suggesting that the anti-proliferative and pro-apoptotic effects of FTY720 are independent of its phosphorylation and interaction with the S1PR1 receptor. We found that activation of S1PR1 through the specific agonist SEW2871 (10μM), FTY720-P (2.5μM), or sphingosine-1-phosphate (100nM) markedly suppresses (~60% inhibition) rather than activates PP2A in normal myeloid progenitors. As expected, knockdown of S1PR1 had no effect on FTY720-mediated PP2A activation in Jak2V617F-transformed cells. Mechanistically we found that Jak2V617F and PP2Ac were found in a ternary complex with the PP2A inhibitor SET. SET knockdown by shRNA restored PP2A activity in Jak2V617F+ Ba/F3 cells to levels similar to those found in non-transformed cells, and led to an 84% decrease in Jak2V617F+-driven colony formation. In addition, co-immunoprecipitation assays revealed that FTY720 (10μM) disrupts Jak2-PP2A, PP2A-SET and Jak2-SET interactions, suggesting that SET may be the target of FTY720. Consistently, affinity chromatography showed that FTY720 efficiently interferes with the ability of C6-ceramide (10μM) to bind SET as the amount of SET eluted from the biotin-labeled C6-ceramide was significantly reduced by exposure of the cell lysate to FTY720. As well, lentiviral-mediated expression of wild type or K209D SET mutant (ceramide binding deficient) in Ba/F3 cells impaired PP2A activity (≥80% decrease), which could be totally rescued by FTY720 only in cells transduced with wild type but not K209D SET. The formal demonstration that FTY720 activates PP2A by displacing SET came when we found SET in anti-NBD immunoprecipitates from Jak2V617F-expressing Ba/F3 cells treated with FTY720-phenoxy-NBD (10μM; 30 min). Together, our data show that FTY720 has the potential to be an effective therapeutic agent for MPD patients by virtue of its low toxicity and ability to activate PP2A by displacing SET; however, FTY720 still retains the ability to become phosphorylated and inhibit, at least in part, PP2A. Thus, we developed non-phosphorylatable FTY720 derivatives and assessed them for their ability to: activate PP2A; induce downregulation/inactivation of targeted kinases (e.g. Jak2, BCR-ABL1, Akt); act as anti-proliferative and pro-apoptotic agent to leukemic but not normal myeloid/lymphoid progenitors; do not interact with S1PR1; and show no in vivo effects on B220+/CD19+ and CD4 or CD8 cellular compartments. These FTY720 derivatives were found to be not immunosuppressive but able to mirror FTY720 in terms of inducing Jak2V617F downregulation and cell killing while retaining the parent compound's minimal toxicity towards untransformed cells. Disclosures: Verstovsek: Incyte Corporation: Research Funding.

Abstract 1222 The molecular mechanism leading to disease progression of chronic myelogenous leukemia (CML) still remains to be identified although enhanced BCR/ABL expression and activity seems to play an important role in controlling genomic stability, differentiation, and self renewal of the leukemic cell clone undergoing blastic tranformation by affecting expression and function of RNA binding proteins (RBPs) like hnRNP A1, hnRNP E2, and hnRNP K (Perrotti D. et al. J. Clin Invest. 2010). We previously reported (Harb J. et al., ASH 2009) that a BCR-ABL1 dosage-dependence and hierarchical organization exists for the expression of hnRNP A1, hnRNP E2, and hnRNP K in cell line models of CML. In fact, as BCR/ABL levels increase, upregulated expression of hnRNP A1 is observed, followed by increased expression of hnRNP E2 and hnRNP K. HnRNP A1 and hnRNP K were also temporally expressed within Lineage- Sca-1+ c-kit+ (LSK), common myeloid progenitors (CMP), and granulocyte macrophage progenitors (GMP) in a mouse model (SCLtTA TRE-BCR/ABL) of chronic myeloid leukemia. Interestingly, hnRNP A1 and hnRNP K levels in BCR/ABL+ mouse progenitors correlated with disease severity as mice with higher levels of these RBPs presented a more progressed phenotype characterized by increased mixed lineage B220+/Mac-1+ progenitors in bone marrow and spleen when compared with mice that developed a CML-CP-like phenotype. Here we show that hnRNP A1, hnRNP E2, and hnRNP K expression levels, as well as BCR/ABL activity are different in HSC (CD34+/CD38-), CMP (CD34+/CD38+/CD45+/IL-3Ra-), and GMP (CD34+/CD38+/CD45+/IL-3Ra+) isolated from peripheral blood of patients in chronic phase (CML-CP) at diagnosis, untreated accelerated phase (CML-AP), and blast crisis (CML-BC). Interestingly, in CML-CP, the highest expression of hnRNP A1 was found in the CD34+/CD38- stem cell fraction and it gradually decreased in the more mature CMP and GMP progenitors (55% and 65% lower, respectively). By contrast, consistent with the role of hnRNP A1 as regulator of progenitor cell proliferation and survival, hnRNP A1 expression progressively increased in the HSC, CMP and GMP fractions isolated from patients in CML-AP and CML-BC. Unlike hnRNP A1, hnRNP E2 and hnRNP K were barely detected in the CD34+/CD38- from CML-CP patients but their expression was markedly pronounced in the HSC fraction of progressed CML patients. In agreement with our cell line data, expression of hnRNP A1 and hnRNP E2 in advanced CML (CML-AP and CML-BC) increases when CD34+/CD38- stem cells undergo maturation toward CMP. Conversely, it appears that hnRNP K expression is the last to increase in CML-BC, suggesting a hierarchical regulation of RBP expression during differentiation and lineage commitment. Expectedly, levels of hnRNP A1 in CMPs increase during disease progression (CP Taken together, these data further implicate RBPs hnRNP A1, hnRNP E2, and hnRNP K in CML disease progression and suggest their possible role in the control of survival of stem and primitive CML-CP progenitors. Disclosures: No relevant conflicts of interest to declare.

Abstract 2901 FTY720 is a sphingosine analog proposed by the FDA for treating Multiple Sclerosis patients because of its immunosuppressive activity, which depends on its ability to prevent lymphocyte egress into the peripheral blood. To act as an immunosuppressive drug, FTY720 undergoes sphingosine kinase 2 (SPHK2) phosphorylation and internalization upon interaction with the sphingosine-1-phosphate receptor 1 (S1PR1). FTY720 also acts as a potent activator of protein phosphatase 2A (PP2A), a tumor suppressor found inactivated in chronic and blast crisis CML with wild type or imatinib/dasatinib-resistant BCR-ABL1, Ph+ B-ALL, KitD816V AML, Jak2V617F+ MPDs and other leukemias/lymphomas. FTY720 treatment of cell lines and primary progenitors isolated from bone marrow of patients with these malignancies, markedly suppressed leukemic cell proliferation/survival and induced apoptosis in a PP2A-dependent manner. Notably, long-term treatment with FTY720 of mice carrying these hematopoietic malignancies significantly prolonged survival and restored normal myelopoiesis without exerting any toxic effect in hematopoietic and non-hematopoietic organs. However, in vivo administration of FTY720 strongly, albeit reversibly, decreases the number of circulating B and T lymphocytes. Here we report that a synthetically phosphorylated FTY720 (FTY720-P) is unable to induce neither PP2A activation nor apoptosis of BCR-ABL1-, Jak2V617F-, or KitD816V-expressing myeloid precursors, indicating that FTY720 phosphorylation is dispensable for its anti-leukemic activity. Thus, we functionally characterize two FTY720 derivatives, QC-FTYSM and OSU-2S, which were synthesized as molecules unable to undergo SPHK2 phosphorylation. Treatment (2.5 uM; 24h) of FTY720-sensitive 32D-BCR/ABL cells with QC-FTYSM and OSU2S results in ∼80% and 40%, respectively, more efficient suppression of BCR-ABL1 expression and kinase activity than that observed with FTY720. Moreover, QC-FTYSM, OSU-2S and FTY720 (2.5uM; 0–60h) induce a progressive block of proliferation and marked induction of apoptosis of 32D-BCR/ABL cells. In fact, a 96%, 98%, and 79% decrease in viability is observed after treatment with QC-FTYSM, OSU-2S and FTY720, respectively. Notably, viability of non-transformed myeloid 32Dcl3 cells is not significantly affected by treatment with FTY720 or its derivatives. Consistent with the ability of FTY720 to induce apoptosis through rescue of PP2A activity, phosphatase assays show identical ability of FTY720, QC-FTYSM and OSU-2S to restore PP2A functionality. In fact, comparable and marked decrease in the amount of inactive Y307-phosphorylated PP2Ac was detected in 32D-BCR/ABL cells treated with FTY720 or its derivatives. To formally demonstrate that QC-FTYSM and OSU-2S lack immunosuppressive activity, we first assessed their ability to be internalized upon interaction/association with the S1PR1 receptor. Thus, cells were transduced with a GFP-tagged S1PR1 and treated with either QC-FTYSM, OSU-2S, or, as positive control, FTY720-P. Confocal microscopy revealed that treatment FTY720-P resulted in a strong S1PR1 internalization. Conversely, exposure of the cells to QC-FTYSM and OSU-2S did not alter the S1PR1 membrane localization, indicating that these molecules did not undergo SHPK2 phosphorylation. Further demonstration of the inability of these compounds to act as immunosuppressive molecules was gained upon in vivo administration of a single dose of FTY720, QC-FTYSM or OSU-2C (10 mg/kg) to wild type FVB/N mice. As expected, percentage of B220+/CD19+ circulating B-cells decreased of ∼90% in FTY720-treated animals. Conversely, the percentage of B-cells after exposure to QC-FTYSM and OSU-2S remained unchanged (≤ 1% decrease). Likewise, the number or CD4+ and CD8+ cells also was not affect by treatment with the QC-FTYSM compound. Note that effect of OSU-2S on T-cells and the toxicity profile and anti-leukemic activity of these drugs in healthy animals and mouse models of deadly leukemias (e.g. T315I+ and blast crisis CML and Ph+ ALL) as well as Ph− MPDs are currently being assessed. Altogether our data indicate that QC-FTYSM and OSU-2S represent two potentially powerful and safe drugs which could be introduced in the current therapeutic protocols for different types of hematopoietic and non-hematopoietic malignancies characterized by functional inactivation of the PP2A tumor suppressor. Disclosures: No relevant conflicts of interest to declare.

MicroRNAs (miRs) and heterogeneous ribonucleoproteins (hnRNPs) are post-transcriptional gene regulators that bind to mRNA in a sequence-specific manner. We showed that hnRNP-E2 inhibits myeloid maturation of bone marrow (BM) progenitors from chronic myelogenous leukemia patients in myeloid blast crisis (CML-BC) by suppressing CEBPA mRNA translation. We report here that loss of miR-328 is induced by BCR/ABL and specifically occurs in CML-BC, and its restored expression rescues differentiation and impairs clonogenic potential of BCR/ABL+ BM progenitors. Accordingly, miR-328 increases during granulocytic differentiation of human CD34+ and mouse LSK BM stem/progenitor cells. Mechanistically, BCR/ABL uses the MAPK-hnRNP-E2 pathway to suppress C/EBPα and miR-328 expression as pharmacologic inhibition of and/or shRNAs against these molecules efficiently restore miR-328 expression. Interestingly, two functional C/EBPα binding sites are present in the miR-328 promoter and positively regulate its transcription. We also show that maturation of differentiation-arrested BCR/ABL+ blasts requires direct interaction of hnRNP-E2 with the miR-328 C-rich regions. Moreover, imatinib treatment restores miR-328 expression, thus allowing its direct binding to hnRNP E2 independent from the RISC complex. Importantly, physiological miR-328 expression decreased hnRNP E2 binding to the uORF/spacer region of endogenous CEBPA mRNA (decoy activity). This, in turn, releases CEBPA mRNA from hnRNP E2 translation inhibition and allows in vitro and in vivo BCR/ABL+ cell differentiation. Although hnRNP E2 was not found in complex with the basic RISC components in BCR/ABL+ cells, miR-328 was found associated to Dicer and Ago2, suggesting that miR-328 also acts through base-pairing with the 3′UTR of mRNA targets in a RISC-dependent manner. In fact, miR-328 suppresses PIM1 protein but not mRNA expression and this effect requires the integrity of the PIM1 3′UTR. Indeed, forced expression of a wild type, but not a kinase-deficient, PIM1 lacking the 3′UTR into miR-328-expressing cells fully rescues BCR/ABL clonogenicity, suggesting that miR-328-induced inhibition of PIM1 accounts for reduced survival of miR-328-infected CML-BCCD34+ blasts. To demonstrate that miR-328 acts on PIM1 in a RISC-dependent manner, we mutated the miR-328 in the seed sequence (miR-328-Mut) while retaining its C-rich character. As expected, miR-328-Mut interacted with hnRNP-E2 and rescued C/EBPα-mediated differentiation, but did not silence PIM1 expression. Thus, the discovery of dual activities for miR-328 which affect myeloid differentiation and survival not only adds a layer to the complexity of mechanisms regulating CML-BC but also highlights the ability of miRNAs to alter mRNA metabolism by acting as molecular decoys for RNA binding proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1950.

Abstract 3259 Poster Board III-1 We have shown that the sphingosine analogue FTY720 markedly induces apoptosis of Ph(+) CML (chronic phase and blast crisis) and B-ALL progenitors regardless of their sensitivity to tyrosine kinase inhibitors through the reactivation of the tumor suppressor PP2A (protein phosphatase 2A). Here we report the identification of the molecular mechanism underlying the ability of FTY720 to activate PP2A, and its therapeutic potential towards Ph(+) and Ph(−) myeloproliferative disorders (MPDs). First, we show that the pro-apoptotic and anti-proliferative effect of FTY720 is not limited to BCR/ABL+ leukemias but can be extended to Jak2V617F-driven MPDs that are also characterized by the extensive (80% inhibition) Jak2V617F-mediated suppression of PP2A activity. In fact, shRNA-mediated interference with the hnRNP A1-SET inhibitory pathway in Jak2V617F-expressing erythro-myeloid precursors revealed that Jak2V617F, like BCR/ABL, utilizes these effectors to impair PP2A function in a dose- and kinase-dependent manner. FTY720 treatment (0.5-1mM) fully restored PP2A activity and resulted in decreased BFU-E colony formation of Jak2V617F-transduced Lin- mouse marrow progenitors. Likewise, FTY720 decreased Jak2V617F expression/activity and inhibited clonogenicity of Jak2V617F-expressing 32D-EpoR+, Ba/F3 and TF-1 cells in PP2A/SHP-1-dependent manner. Secondly, we show that PP2A is also inhibited in a SET-dependent manner in CD34+/CD38- stem cells (HSCs) from CML patients. In Ph(+) HSCs, FTY720 but not imatinib or dasatinib decreases the number of long-term culture initiating cells (LTC-IC) and quiescent stem cells (CFSEmax) through activation of BCR/ABL-independent caspase-mediated apoptosis. Importantly, enhanced self-renewal of Ph(+) HSCs seems to depend on constitutive nuclear β-catenin transcriptional activity. In fact, FTY720 or PP2Ac lentiviral transduction, but not imatinib/dasatinib, induces b-catenin inactivation/degradation as measured by in situ immunofluorescence and LET/TCF assays. The effect of FTY720 in Ph(+) HSCs relies on the PP2A ability to override the BCR/ABL-independent inactivation of the β-catenin negative regulator GSK-3β, as treatment with the GSK-3β inhibitors LiCl and SB216763 increased the CFSEmax fraction and hampered the pro-apoptotic effect of FTY720 on quiescent Ph(+) HSCs. Notably, FTY720 did not affect normal stem/progenitor cell viability. Similar results were obtained with the LSK fraction from SCL-tTA/BCR/ABL mice. Reportedly, FTY720 to act as an immunosuppressant requires phosphorylation by sphingosine kinase (SPHK) 2. To determine whether conversion of FTY720 into FTY720-P is important for its anti-leukemic activity in BCR/ABL- and Jak2V617F-expressing cells, we used growth factor-dependent hematopoietic cells transformed by the constitutive activity of these oncogenic tyrosine kinases. In Jak2V617F- and BCR/ABL-transformed cells, PP2A activation by FTY720 (2.5μM, 6h) is not changed when phosphorylation is prevented by treatment with the SPHK inhibitor dimethylsphingosine (2.5μM, 6h). Accordingly, FTY720-P did not activate PP2A and did not affect BCR/ABL- and Jak2V617F-driven colony formation, indicating that the anti-leukemic activity of FTY720 does not require its phosphorylation. Because we have shown that sphingolipid PP2A activator ceramide specifically binds to SET, thus disrupting the SET/PP2A interaction in non hematopoietic cells, we investigated the effect of BCR/ABL or Jak2 V617F and that of FTY720 on ceramide production. LS-MS mass-spectrometry showed that levels of ceramide were similar in untreated and imatinib-treated BCR/ABL+ cells. Similarly, levels of diacylglycerol (DAG) kinase-phosphorylated ceramide were unchanged by treatment with FTY720, suggesting that oncogenic tyrosine kinase-induced suppression of PP2A and FTY720-dependent PP2A reactivation are not ceramide-mediated. Accordingly, FTY720 displaces biotin-labeled C6-ceramide (10μM) from SET, as measured by ceramide affinity chromatography followed by determination of SET levels in the eluted fraction. Thus, FTY720 represents a powerful therapeutic tool as it has the potential to treat and, perhaps, eradicate Ph(+) and Ph(−) MPDs by efficiently inhibiting oncogene-dependent and -;independent signals through the reactivation of the tumor suppressor PP2A via disruption of the SET/PP2A inhibitory complex. Disclosures: Cortes: Novartis: Research Funding. Cancelas:CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

Abstract 855 MicroRNAs (miRs) and heterogeneous ribonucleoproteins (hnRNPs) are post-transcriptional gene regulators that bind mRNA in a sequence-specific manner. We have reported that a) hnRNP-E2 suppresses CEBPA mRNA translation and inhibits myeloid maturation of bone marrow (BM) progenitors from chronic myelogenous leukemia patients in myeloid blast crisis (CML-BCCD34+; Perrotti et al, Nat Genet 2002); and b) miR-328 expression is lost in myeloid CML-BCCD34+ progenitors (n=6) and its restored expression at physiological levels rescues granulocytic differentiation and impairs clonogenic potential of primary BCR/ABL+ blasts (Eiring et al, ASH 2007). Here we show by Northern blot, real-time PCR, and microarray analyses that miR-328 levels increase during granulocytic differentiation of normal human CD34+ and mouse Lin− BM progenitors, but not during differentiation towards erythroid, megakaryocytic or monocytic lineages. BCR/ABL uses the same MAPKERK1/2-hnRNP-E2 signaling pathway to suppress both C/EBPα and miR-328, as pharmacologic or shRNA-mediated inhibition of these molecules restored miR-328 expression in BCR/ABL+ cells. In fact, two functional C/EBPα binding sites are present in the miR-328 promoter region and C/EBPα interacts in vivo with these regulatory elements to enhance miR-328 transcription. Importantly, we also show that restored maturation of BCR/ABL+ blasts requires direct interaction of hnRNP-E2 with the C-rich regions of miR-328. Indeed, RNA-immunoprecipitation (RIP) assays demonstrated that miR-328 directly binds to hnRNP-E2 independent of the RNA-induced silencing complex (RISC). Furthermore, ectopic miR-328, but not miR-181b, resulted in decreased in vivo binding of hnRNP-E2 to the uORF/spacer region of CEBPA mRNA, thereby releasing CEBPA from hnRNP-E2 translation inhibition and rescuing C/EBPa-driven neutrophil maturation (decoy activity). Differentiation of miR-328-expressing CML-BCCD34+ blasts (88.8±2.4% post-mitotic cells) correlated with induction of C/EBPa protein expression, whereas CEBPA mRNA and hnRNP E2 protein levels remained unchanged. The existence of a direct miR-328/hnRNP-E2/CEBPA interplay was formally demonstrated in vitro using RRL-directed translation assays and in vivo using the 6.15 clone of 32D-BCR/ABL cells that do not express endogenous CEBPA mRNA and require ectopic C/EBPα (wt-uORF-CEBPA) for differentiation. Addition of miR-328, but not miR-330, to hnRNP-E2-containing RRL reactions increased newly synthesized 35S-C/EBPa levels by >100%. Likewise, forced miR-328 expression in vivo resulted in decreased hnRNP-E2 binding to CEBPA mRNA, induction of C/EBPa protein but not mRNA and rescued granulocytic differentiation of 6.15-wt-uORF-CEBPA but not vector-transduced 6.15 cells. While hnRNP-E2 was not found in complex with basic RISC components (Dicer, TRBP2 and Ago2), RIP assays detected miR-328 associated to Dicer and Ago2 in miR-328-expressing cells, suggesting that it also acts through canonical RISC-dependent base-pairing with mRNA targets. Indeed, we identified the BCR/ABL-regulated PIM1 serine-threonine kinase as a bona fide miR-328 target in BCR/ABL+ cells. Ectopic miR-328 suppressed PIM1 protein but not mRNA levels, and this effect required integrity of the miR-328 binding site present in the PIM1 3'UTR. Forced expression of a wild-type but not kinase-deficient PIM1 lacking the 3'UTR into miR-328-expressing cells fully rescued BCR/ABL clonogenicity, suggesting that miR-328-induced PIM1 suppression accounts for reduced survival of miR-328-infected BCR/ABL+ blasts. To show that miR-328 acts on PIM1 in a RISC-dependent manner, we mutated the miR-328 seed sequence (miR-328-Mut) while retaining its C-rich character. Similar to wild-type miR-328, miR-328-Mut efficiently interacted with hnRNP-E2, restored C/EBPa protein expression and rescued granulocytic differentiation, but was unable to silence PIM1 in 32D-BCR/ABL cells, indicating that the C-rich character of miR-328 is essential for its decoy activity, while its seed sequence integrity is necessary for RISC-dependent pairing to mRNA targets. Thus, the discovery of dual activities for miR-328 not only adds a new layer of complexity to the mechanisms regulating CML disease progression, but also highlights the ability of miRNAs to alter mRNA metabolism by acting as molecular decoys for RNA-binding proteins. Disclosures: Cortes: Novartis: Research Funding.