ALBERT

Description: Abstract 2885 MicroRNAs (miRs) are involved in the initiation, progression and dissemination of CLL cells (Calin GA, Croce CM. Blood 114:4761, 2009). Recent studies showed that high levels of miR-155, previously shown to regulate hematopoietic cell development, are expressed in CLL cells. Because transgenic miR-155 overexpression in the mouse stimulates B-cell proliferation, it is thought that miR-155 plays a role in the pathogenesis of CLL (Calin GA et al. N Engl J Med 353:1793, 2005). STAT3 is constitutively activated in CLL and induces the transcription of several STAT3-regulated genes. A recent study demonstrated that STAT3 activates miR-21 and miR-181b-1 (Iliopolus D. et al. Mol Cell 39:493, 2010). Therefor we wondered whether STAT3 enhances the expression of miR-155 in CLL cells. Because a sequence analysis revealed that the promoter of miRNA-155 harbors γ-interferon activation sequence-like elements typically activated by STAT3, we sought to determine whether STAT3 directly activates miR-155 expression. We generated truncated constructs of the miR-155 promoter, co-transfected them into MM1 cells together with STAT3 small interfering (si) RNA (siRNA), and assessed their luciferase activity. The luciferase activity data suggested that of the two putative STAT3 binding sites only one site is involved in STAT3 induced transcription because STATR3-siRNA reduced the activity of miRNA-155 promoter of constructs that harbor this site. To confirm these data we performed an electrophoretic mobility shift assay (EMSA) and chromatin immune-precipitation (ChIP). The EMSA confirmed that STAT3 bound the miR-155 promoter in fresh CLL cells, and ChIP confirmed that STAT3 bound one putative STAT3-binding site in the miR-155 promoter but not to the other, as demonstrated by the luciferase assay; STAT3 co-immuno-precipitated only one putative STAT3 binding region of miR-155 promoter and other STAT3-regulated genes. Finally, STAT3-small hairpin RNA (shRNA) downregulated miR-155 and other STAT3-regulated genes, suggesting that constitutively activated STAT3, binds miR-155 promoter and induces miR-155 transcription in CLL cells. Disclosures: Keating: Celgene Corporation: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Xcenda: Consultancy, Speakers Bureau.

Description: Abstract 2886 Non-coding RNAs regulate the expression of more than 30% of protein-coding genes both at a post-transcriptional and translational level. Although approximately 1000 microRNAs (miRs) have been identified in the human genome, little is known about the mechanisms that regulate miR expression. STAT3 regulates the transcription of miR-21, and miR181b-1, binds to their promoter and induce neoplastic cell transformation (Iliopoulos, Jaeger et al. 2010). Because STAT3 is constitutively activated in CLL cells (Hazan-Halevy, Harris et al. 2010) we sought to investigate how STAT3 affects non-coding RNA gene expression in CLL cells. We transfected peripheral blood CLL cells from 3 different patients with STAT3-shRNA and assessed non-coding RNA levels using a non-coding RNA array containing 2277 human miR probes, 960 from ultra-conserved genes and 3540 of long non-coding RNAs. When compared to transfection control, 152 probes from 78 non-coding RNA genes were differentially expressed (134 down-regulated and 18 up-regulated), suggesting that STAT3 affects the non-coding RNA network in CLL cells. Supervised clustering analysis was used to select genes for validation. By using quantitative RT-PCR we validated our gene array analysis. Similar to the data obtained by the non-coding RNA array, we found that transfection of CLL cells with STAT3- down-regulated the levels of miR-21, miR-155, and miR-320b. Binding site prediction programs and ChIP-seq data embedded in the UCSC genome browser determined that in 5 of 7 genes, down-regulated by STAT3-shRNA transfection, were either putative or experimentally confirmed STAT3-binding sites, indication that STAT3 directly regulates the transcription of those miRs. It has been shown that the interaction between miRs and single stranded RNA is dependent on base pairing in a seed region at positions 2 to 8. High levels of 4.8kb single stranded STAT3 RNA transcripts, present in CLL cells, provide a substrate for such paring. Therefore, we assumed that STAT3 functions as a “RNA sponge” soaking up miRs and altering their effective levels and function. To test this hypothesis we used the pattern-based RNA22 algorithm and identified potential miR targets. We than calculated the energy that would be released if the corresponding RNA/RNA complexes are saturated. We found that the energy released from binding of miRs to STAT3 sequences would be higher than energy released from binding to a random sequence with same length and base content suggesting that STAT3 “sponges out” miRs in a sequence specific manner. Thus, CLL cells are characterized by an ongoing interaction between STAT3-mediated transcriptional regulation of non-coding RNA and miR-mediated translational regulation of coding genes. Disclosures: No relevant conflicts of interest to declare.

Description: The metabolic profile of mammalian cells is determined primarily by the cells’ proliferation rate. Unlike circulating memory B cells, which are typically quiescent and proliferate only in response to external stimuli, approximately 1% of chronic lymphocytic leukemia (CLL) cells proliferate daily. We sought to determine how CLL cells adjust their metabolism to meet increased energy demands imposed by their proliferation rate. Muscle cells, which proliferate at rates similar to those of CLL cells, preferentially use intracellular stored triglycerides as an available energy source. Similar to muscle cells, CLL cells express lipoprotein lipase (LPL), an enzyme that catalyzes the hydrolysis of tryglycerides into free fatty acids (FFA). We wondered whether CLL cells use a similar pathway. In reviewing bone marrow biopsies of patients with CLL, we identified clear-appearing oil red O-positive vacuoles in the cytoplasm of CLL cells. Using electron microscopy, we confirmed that these lipid vacuoles were present in 95% of CLL peripheral blood cells but not in normal B cells. To determine whether CLL cells metabolize FFA, we incubated CLL cells with or without FFA (palmitate or oleate) in a sealed flask and measured the dissolved O2 (dO2) content in the medium of the cultured cells after 48 h. Compared with CLL cells incubated in the absence of FFA, dO2 levels were significantly reduced when FFA was added. In contrast, dO2 levels were not reduced after FFA was added to cultures of normal B lymphocytes, suggesting that unlike normal B cells, CLL cells acquired the capacity to metabolize FFA. Transfecting CLL cells with LPL small interfering RNA abrogated the capacity of CLL cells to metabolize FFA, suggesting that FFA metabolism in CLL cells is LPL dependent. We and other groups found that LPL is abundantly expressed in CLL cells. Because STAT3 is constitutively activated in CLL cells and because we identified putative STAT3 binding sites in the LPL promoter, we hypothesized that STAT3 induces aberrant expression of LPL in CLL cells. By transfecting a luciferase reporter gene driven by LPL promoter fragments into MM1 cells, we found that STAT3 activates the LPL promoter, and by using chromatin immunoprecipitation and electrophoretic mobility shift assays, we confirmed that STAT3 binds to the LPL promoter in MM1 and in CLL cells. To confirm these data, we transfected CLL cells with a lentiviral STAT3 short hairpin RNA. Unlike the empty lentiviral vector, STAT3–small interfering RNA downregulated mRNA levels of LPL and several STAT3 target genes and downregulated LPL protein levels. Taken together, our data suggest that CLL cells store lipids in cytoplasmic vacuoles, produce LPL, and adapt their metabolism to utilize intracellular stored lipids for energy production, a process that is driven by constitutively activated STAT3. Disclosures O'Brien: Amgen, Celgene, GSK: Consultancy; CLL Global Research Foundation: Membership on an entity's Board of Directors or advisory committees; Emergent, Genentech, Gilead, Infinity, Pharmacyclics, Spectrum: Consultancy, Research Funding; MorphoSys, Acerta, TG Therapeutics: Research Funding.

Description: Abstract 1742 Primary myelofibrosis (PMF) is a stem cell–derived hematologic malignancy, characterized by an expansion of one or more myeloid lineage resulting in bone marrow (BM) hypercellularity, magakaryocyte proliferation with atypia, granulocytic proliferation, and reticulin and/or collagen fibrosis. An acquired activating mutation in Janus kinase 2 at codon V617F (JAK2V617F) is detected in BM cells of the majority of patients with PMF. Constitutively activated JAK2 induces phosphorylation and activation of STAT3. Phosphorylated STAT3 forms heterodimers, translocates to the nucleus, binds to DNA, activates STAT3-target genes, and induces production of cytokines that interact with the BM microenvironment. Hematopoietic stroma derived soluble factors provide PMF cells with survival advantage (Manshouri et al. Cancer Res 71: 3831, 2011) and, as reported previously, most of these factors activate NF-κB in a variety of cell types. NF-κB plays an important role in the survival and proliferation of normal and neoplastic cells. In several hematologic malignancies, the NF-κB p65/p50 dimers were found to be activated to variable degrees. The activation of NF-κB is mediated by either the canonical pathway or the alternative pathway. The canonical pathway is typically activated by extracellular signals that activate the β subunit of the IκB kinase (IKK) complex (IKKβ) that induces the phosphorylation and degradation of the NF-κB inhibitor IκBα. Following IκBα degradation, NF-κB heterodimers translocate to the nucleus and bind to DNA. We have recently found that in chronic lymphocytic leukemia (CLL) constitutively activated STAT3 induces the production of unphsophorylated (U) STAT3. U-STAT3 binds to the NF-κB dimers p65/p50 in competition with IκB and the U-STAT3/NF-κB complex shuttles to the nucleus where NF-κB binds to DNA and activates NF-κB-regulated genes (Liu et al. Mol Cancer Res 9: 507, 2011). Because in PMF constitutively activated JAK2 induces phosphorylation of STAT3 and this activated form of STAT3 induces the production of U-STAT3, we wondered whether, like in CLL, U-STAT3 activates NF-κB in PMF. To determine whether NF-κB is constitutively activated in PMF we obtained BM low density cells from untreated patients with PMF. First we studied low-density BM cells of 11 patients with PMF using the electrophoretic mobility shift assay (EMSA). Cells of all samples bound to a p65/NF-κB DNA-labeled probe and the addition of an unlabelled (cold) p65/NF-κB probe attenuated or completely eliminated the binding. Typically, NF-κB-DNA binding appears and disappears due to repeated degradation and re-synthesis of IκB and the consequent activation and inactivation of NF-κB, respectively. Because we found that NF-κB is constitutively activated in all PMF BM samples we hypothesized that, like in CLL cells, activation of NF-κB in PMF cells is induced by an IκB-unrelated mechanism as reported by Yang J et al. (Cancer Res 65:939, 2005). By using immunoprecipitation of two different PMF BM samples we determined that STAT3 binds to the RelA/p65 NF-κB protein, and by using EMSA we found that anti-STAT3, similar to anti- NF-κB p65 antibodies, attenuated the binding of PMF BM cell extract to the NF-κB DNA probe. Taken together, our data suggest that U-STAT3 binds the NF-κB dimers p65/p50 and constitutively activates NF-κB in PMF. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3879 GM-CSF stimulates proliferation of granulocytes, macrophages, and hematopoietic progenitors. Upon binding to its cellular receptor (R), GM-CSF induces dimerization of the GM-CSFR α and β subunits, phosphorylation of Janus kinase (JAK)-2, and activation of downstream signaling pathways. Because GM-CSF improved the response to Rituximab monotherapy in patients with CLL (Ferrajoli A. Leuk Lymphoma 50:514, 2009) and was found to upregulate CD20 cell surface antigen expression (Vanugopal P. et al. Leuk Res 24:411, 2000), we investigated the effect of GM-CSF on CLL cells. Incubation of peripheral blood (PB) CLL cells with increasing concentrations of GM-CSF (0.05 to 1.0 μM) did not induce the phosphorylation of STAT3, STAT5, AKT, or ERK as assessed by western immunoblot, or phosphorylation of JAK2 as assessed by immunoprecipitation. Therefore, we investigated whether CLL cells express GM-CSFR. Western immunoblot studies revealed that, like normal B lymphocytes, CLL cells do not express GM-CSFRβ but, unlike normal B cells, CLL cells express high levels of GM-CSFRα. Flow cytometry analysis of PB cells from 8 patients with CLL showed that 13 to 59% of CLL cells (CD19+/CD5+) co-expressed CD116 (GM-CSFRα) but not CD131 (GM-CSFRβ). Thus, we wondered what induces GM-CSFRα expression in CLL cells. Because STAT3 is constitutively activated in CLL and sequence analysis revealed that the GM-CSFRα promoter harbors γ-interferon activation sequence (GAS)-like elements typically activated by STAT3, we sought to determine whether STAT3 activates GM-CSFRα. In MM1 cells, interleukin (IL)-6 induced STAT3 phosphorylation and up-regulated GM-CSFRα whereas STAT3-siRNA down-regulated both STAT3 and GM-CSFRα protein levels, suggesting that STAT3 activates transcription of GM-CSFRα. To clarify these findings, we cloned the human GM-CSFRα promoter, generated a series of truncated promoter constructs and assessed their activity using a luciferase assay. We found that IL-6 augmented luciferase activity of GM-CSFRα promoter −4012 – +23, −3018 – +23, −2517 – +23, and −496 – +23, suggesting that IL-6 enhanced GM-CSFRα expression by activating STAT3. Furthermore, we established that regions, located between bp −3581 TTGTTGAAAA −3572, −2984 TTTTCTTAA −2976 and −77 TTTCCCAA −70, harbor GAS-like elements that activated the GM-CSFRα promoter upon exposure to IL-6. Binding of STAT3 to those regions in IL-6-stimulated MM1 cells was confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). To test whether STAT3 induced transcription of GM-CSFRα in CLL, we obtained fresh PB CLL cells and, by using the same GAS-like element-containing probes, performed EMSA. CLL cell nuclear protein bound these probes and anti-STAT3 and -phosphoserine STAT3 antibodies attenuated the binding. CLL cell ChIP confirmed that STAT3 binds to the promoter of GM-CSFRα as well as the promoters of the STAT3-regulated genes STAT3, c-Myc and P21, but not to that of the control gene RPL30. Finally, using qRT-PCR and western blot analysis we determined that STAT3-shRNA down-regulated GM-CSFRα, STAT3 and STAT3-regulated gene mRNAs, and STAT3 and GM-CSFRα protein levels. Taken together, these data suggest that constitutively activated STAT3 binds to the GM-CSFRα promoter, activates its transcription, and induces production of GM-CSFRα protein in CLL cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3910 Introduction: For several decades CLL has been defined as a chronic leukemia characterized by a passive accumulation of small neoplastic lymphocytes that do not proliferate and do not die. This definition has been revised in recent years as it was shown that CLL cells do proliferate mostly in proliferation centers. Because the increase in peripheral blood (PB) CLL cell count was lower than their proliferation rate, it was intuitively assumed that proliferation of CLL cells is accompanied by spontaneous apoptosis, and as proliferation rate increases so does the apoptosis rate. Because STAT3 is constitutively activated in CLL cells and provides CLL cells with survival advantage (Hazan-Halevy I. et al. Blood 115:2852, 2010), we wondered whether decreased levels of intracellular STAT3 would correlate with increased apoptosis of CLL cell. Methods and Results: Assessment of apoptosis rate by flow cytometry using propidium iodide (PI) and annexin V staining demonstrated that a significant fraction of freshly obtained PB CLL cells undergo spontaneous apoptosis in samples of 4 out of 4 patients with CLL. Spontaneous apoptosis was detected in 23% of CLL cells from a patient with a white blood cell count (WBC) of 16,000 (*10⋀6/L), in 20% of CLL cells from a patient with a WBC of 32,800, in 41 % of CLL cells from a patient with a WBC of 55,600, and in 65% of CLL cells from patient with a WBC of 101,000 (*10⋀6/L), suggesting that spontaneous apoptosis rates correlate with the number of circulating CLL cells. Because apoptotic cells are removed by the reticuloendothelial system, early apoptosis of CLL cells was assessed by quantification of cleaved PARP levels in CLL cells from 36 patients using an enzyme linked immunosorbent assay (ELISA). WBC in half of those patients ranged from 5,000 to 17,000 (*10⋀6/L) (median: 14,500) and in the other half from 151,000 to 680,000 (*10⋀6/L) (median: 237,000). The median level of cleaved PARP was three times higher in cells from patients with a high lymphocyte count than in cells from patients with a low lymphocyte count (P = 0.007), confirming our hypothesis that as disease burden increases so does CLL cell apoptosis rate. Because STAT3 plays a key role in CLL cell survival we sought to determine whether CLL cell apoptosis rates correlate with intracellular STAT3 levels. We quantified STAT3 levels in PB CLL cells from 185 CLL patients using an ELISA. Our data revealed a linear correlation between the number of CLL cells and intracellular STAT3 levels. The higher the lymphocyte counts, the lower were STAT3 levels (rp = 0.28, P