Supplemental Table 1. Patient Characteristics

Alteration in the DNA replication, repair or recombination processes is a highly relevant mechanism of genomic instability. Despite genomic aberrations manifested in hematologic malignancies, such a defect as a source of biomarkers has been underexplored. Here, we investigated the prognostic value of expression of 82 genes involved in DNA replication-repair-recombination in a series of 99 patients with chronic lymphocytic leukemia without detectable 17p deletion or TP53 mutation. We found that expression of the POLN gene, encoding the specialized DNA polymerase ν (Pol ν) correlates with time to relapse after first-line therapy with fludarabine. Moreover, we found that POLN was the only gene up-regulated in primary patients' lymphocytes when exposed in vitro to proliferative and pro-survival stimuli. By using two cell lines that were sequentially established from the same patient during the course of the disease and Pol ν knockout mouse embryonic fibroblasts, we reveal that high relative POLN expression is important for DNA synthesis and cell survival upon fludarabine treatment. These findings suggest that Pol ν could influence therapeutic resistance in chronic lymphocytic leukemia. (Patients' samples were obtained from the CLL 2007 FMP clinical trial registered at: clinicaltrials.gov identifer: 00564512).

The nucleoside analog cytarabine, an inhibitor of DNA replication fork progression that results in DNA damage, is currently used in the treatment of acute myeloid leukemia (AML). We explored the prognostic value of the expression of 72 genes involved in various aspects of DNA replication in a set of 198 AML patients treated by cytarabine-based chemotherapy. We unveiled that high expression of the DNA replication checkpoint gene CHEK1 is a prognostic marker associated with shorter overall, event-free, and relapse-free survivals and determined that the expression of CHEK1 can predict more frequent and earlier postremission relapse. CHEK1 encodes checkpoint kinase 1 (CHK1), which is activated by the kinase ATR when DNA replication is impaired by DNA damage. High abundance of CHK1 in AML patient cells correlated with higher clonogenic ability and more efficient DNA replication fork progression upon cytarabine treatment. Exposing the patient cells with the high abundance of CHK1 to SCH900776, an inhibitor of the kinase activity of CHK1, reduced clonogenic ability and progression of DNA replication in the presence of cytarabine. These results indicated that some AML cells rely on an efficient CHK1-mediated replication stress response for viability and that therapeutic strategies that inhibit CHK1 could extend current cytarabine-based treatments and overcome drug resistance. Furthermore, monitoring CHEK1 expression could be used both as a predictor of outcome and as a marker to select AML patients for CHK1 inhibitor treatments.

In chronic lymphocytic leukemia (CLL), the increment in PBLs is slower than the expected increment calculated from the cells’ proliferation rate, suggesting that cellular proliferation and apoptosis are concurrent. Exploring this phenomenon, we found overexpression of caspase-3, higher cleaved poly (ADP-ribose) polymerase levels (p < 0.007), and a higher apoptosis rate in cells from patients with high counts compared with cells from patients with low counts. Although we previously found that STAT3 protects CLL cells from apoptosis, STAT3 levels were significantly higher in cells from patients with high counts than in cells from patients with low counts. Furthermore, overexpression of STAT3 did not protect the cells. Rather, it upregulated caspase-3 and induced apoptosis. Remarkably, putative STAT3 binding sites were identified in the caspase-3 promoter, and a luciferase assay, chromatin immunoprecipitation, and an EMSA revealed that STAT3 activated caspase-3. However, caspase-3 levels increased only when STAT3 levels were sufficiently high. Using chromatin immunoprecipitation and EMSA, we found that STAT3 binds with low affinity to the caspase-3 promoter, suggesting that at high levels, STAT3 activates proapoptotic mechanisms and induces apoptosis in CLL cells.

Abstract While reviewing chronic lymphocytic leukemia (CLL) bone marrow slides, we identified cytoplasmic lipid vacuoles in CLL cells but not in normal B cells. Because lipoprotein lipase (LPL), which catalyzes hydrolysis of triglycerides into free fatty acids (FFA), is aberrantly expressed in CLL, we investigated whether LPL regulates the oxidative metabolic capacity of CLL cells. We found that unlike normal B cells, CLL cells metabolize FFAs. Because STAT3 is constitutively activated in CLL cells and because we identified putative STAT3 binding sites in the LPL promoter, we sought to determine whether STAT3 drives the aberrant expression of LPL. Transfection of luciferase reporter gene constructs driven by LPL promoter fragments into MM1 cells revealed that STAT3 activates the LPL promoter. In addition, chromatin immunoprecipitation confirmed that STAT3 binds to the LPL promoter. Furthermore, transfection of CLL cells with STAT3-shRNA downregulated LPL transcripts and protein levels, confirming that STAT3 activates the LPL gene. Finally, transfection of CLL cells with LPL-siRNAs decreased the capacity of CLL cells to oxidize FFAs and reduced cell viability. Implications: Our study suggests that CLL cells adopt their metabolism to oxidize FFA. Activated STAT3 induces LPL, which catalyzes the hydrolysis of triglycerides into FFA. Therefore, inhibition of STAT3 is likely to prevent the capacity of CLL cells to utilize FFA. Mol Cancer Res; 13(5); 944–53. ©2015 AACR.

In CLL STAT3 is constitutively phosphorylated on serine 727 residues and activates anti-apoptotic genes. Therefore, we hypothesized that STAT3 levels and rate of spontaneous apoptosis would be inversely correlated. To test levels of STAT3 we used quantitative western immunoblotting and found that levels were significantly higher in patients with high (N = 32) compared to low (N = 32) WBC counts (P = 0.007). To test the rate of spontaneous apoptosis we stained the cells of 19 CLL patients with Annexin V and PI and found that apoptosis rates were highly correlated with white blood cell (WBC) counts (rp=0.88, P< 0.0001). Accordingly, levels of cleaved PARP were 3 times higher in patients with high (N = 32) compared to low (N = 32) WBC counts (P = 0.007). Hence, contrary to our hypothesis in high-count CLLs levels of STAT3 and rate of spontaneous apoptosis are both increased. Intrigued by these findings we wondered whether when present at high levels STAT3 induces apoptosis of CLL cells. To test this theory we first overexpress STAT3 in MM1 cells and found that overexpression of STAT3 upregulated caspase3 levels and induced apoptosis of MM1 cells. Because sequence analysis revealed that the promoter of caspase-3 harbors putative STAT3 binding sites, we sought to determine whether STAT3 activates caspase-3. Chromatin immunoprecipitation (ChIP) and an electrophoretic mobility shift essay (EMSA) confirmed that STAT3 binds the caspase-3 promoter, and a Luciferase assay validated that STAT3 activates the caspase-3 promoter in IL-6-treated MM1 cells. To assess STAT3’s binding affinity to the promoter of caspase-3, we prepared serial dilutions of CLL cell DNA and, using ChIP and EMSA, found that STAT3’s binding affinities to p21 and c-Myc were 8 and 4 times higher than STAT’s binding affinity to caspase-3, suggesting that at high levels STAT3 are required to activate caspase-3. Taken together, these findings suggest that STAT3 has a previously unknown pro-apoptotic function. When present at high levels, STAT3 activates Caspase3 and induces apoptosis rather than providing CLL cells with survival advantage.

Chronic B-cell lymphocytic leukaemia (CLL) is generally an indolent disease, with most patients surviving years without treatment although some progress more rapidly. Several markers, such as IGHV mutational status (Hamblin et al, 1999), cytogenetic abnormalities (Döhner et al, 2000) and recurrent gene mutations (Wang et al, 2011; Jeromin et al, 2014), have helped to better stratify patient risk, but few are routinely used by clinicians and they are still relatively unreliable, reflecting the clinical and physiopathological heterogeneity of the disease. Thus there remains room for improvement and new molecular markers. Our understanding of the various roles of small nucleolar RNAs (snoRNAs) in cancer is continually expanding. Besides their well-known function in ribosomal RNA modifications, snoRNAs have recently been described as new biomarkers in haematological cancers (Ronchetti et al, 2012, 2013; Valleron et al, 2012a,b). Notably, Ronchetti et al (2013) studied a cohort of Binet stage A CLL patients, reporting that a specific snoRNA signature was predictive of outcome for early stage CLL. In this study, we investigated the snoRNA expression profiles in CLL patients fully annotated for both the classical prognostic markers (IGHV mutational status, cytogenetic abnormalities, Binet stage) and recurrent somatic mutations (TP53, NOTCH1, SF3B1). The methods and patient characteristics are described in Data S1, Tables SI and SII. Using high-throughput quantitative polymerase chain reaction (Fluidigm, Les Ullis, France), we first determined whether snoRNA profiling (n = 221, covering more than two-thirds of previously described snoRNAs) could discriminate between CLL prognostic subgroups in an exploration set of 58 treatment-naive CLL and five normal B-cells. Unsupervised hierarchical clustering showed that patients did not cluster together when considering criteria such as IGHV mutational status, Binet stage, age, gender, karyotype, fluorescence in situ hybridization and NOTCH1/TP53/SF3B1mutation, but instead were scattered along the dendrogram (Fig S1). Supervised analysis also did not find a snoRNA signature specific to the conventional clinico-biological parameters, suggesting that snoRNA expression profiles were not associated with the aforementioned factors impacting on CLL outcome. The apparent contradiction between our results and those reported by Ronchetti et al (2013) could be explained by the differences between the two cohorts as we included more cases presenting adverse prognosis factors. However, the unsupervised analysis showed that normal B-cells clustered together, suggesting that snoRNA expression profiles are influenced by leukaemic phenotype. Sparse Partial Least Squares-Discriminant Analysis (sPLS-DA) is a mathematical method that can be used to determine the best gene set while also allowing discrimination between two groups. Thus we used sPLS-DA to identify snoRNAs that could discriminate between normal B-cells and CLL cases and obtained a minimal set of four snoRNAs (Fig 1A, B). SNORD35B, SNORD71, SNORD116-11 and SNORD116-25 were sufficient to separate healthy cells from leukaemic cells in hierarchical clustering analysis (Fig 1C). These results were confirmed in a validation cohort that included 56 new, treatment-naïve, CLL and five new normal B-cells, stressing the robustness of this signature (Fig 1D). We then investigated whether snoRNA profiles could be associated with differences in CLL progression. To this end we focused on treatment-free survival (TFS), which is the first prognostic parameter in the disease course, as some CLL patients remain therapy-free for many years while others rapidly progress and require treatment. The patients included in our exploration and validation cohorts had a median TFS of 34 and 30 months respectively (in keeping with the frequency of adverse biological risk factors in our population). We could not demonstrate any dysregulation of a specific set of snoRNAs based either on the median TFS of the exploration cohort or among the different subtypes, with the exception of the IGHV-mutated (IGHV-M) patients. Most IGHV-M patients display a prolonged TFS but snoRNA expression profiles enabled division into two prognostic subgroups. Using sPLS-DA we identified a set of 20 snoRNAs (Table SIII) that are globally overexpressed among IGHV-M patients with an unexpectedly short TFS (Fig S2). To increase the number of IGHV-M patients, the exploration and validation cohorts were merged to compare TFS between IGHV-M patients with high and low expression of the 20 snoRNAs (according to the signature expression index, Fig S3) and IGHV-unmutated (IGHV-UM) patients. Kaplan–Meier analysis confirmed that overexpression of the 20 snoRNAs was associated with a shorter TFS (median: 32 months) compared to underexpression of the 20 snoRNAs (median: 144 months) (Fig 2A). Addition of IGHV-UM to the Kaplan–Meier analysis indicated that the TFS of IGHV-M patients with high expression of these snoRNAs was similar to that of IGHV-UM patients (Fig 2B), leading us to postulate that IGHV-M patients with greater progression potential could be further classified using snoRNAs. However, due to the low number of IGHV-M cases investigated, this signature needs further validation in a larger prospective set of IGHV-M patients. We then investigated the putative function of these snoRNAs in CLL proliferation. One of the main criteria indicating the need to initiate treatment is increased lymphocyte doubling time (Hallek et al, 2008). Thus, a shorter TFS can be related to increased cell proliferation. Therefore, we treated primary cells from five CLL patients with immunostimulators (interleukin 2 and CpG oligodeoxynucleotides) to induce proliferation (Fig S4A). We also induced the proliferation of normal B-cells using lipopolysaccharide and anti-IgM (Fig S4B) to assess the potential abnormal regulation of snoRNAs in CLL. Following proliferation, quantification showed that 11–17 of the 20 snoRNAs had increased expression in CLL, depending on the patient (Fig S5). Similarly, 15–18 snoRNAs had increased expression in normal B-cells, depending on the donor (Fig S6). This suggests that most of the 20 snoRNAs could be functionally relevant in a proliferation context. Interestingly, comparison of the fold change in expression of the 20 snoRNAs between CLL and normal B-cells shows that only seven were consistently upregulated following proliferation in both normal B-cells and CLL. Two snoRNAs (SNORA80; SNORD1A) were consistently downregulated in CLL (Fig S5). These results indicate that some snoRNAs are deregulated in response to proliferation in CLL. Nevertheless, as these 20 signature snoRNAs are globally upregulated in short TFS IGHV-M patients, our results imply that the signature reflects more than just the proliferation potential of the leukaemic cells. Other factors affecting TFS, such as increased resistance to apoptosis, should also be explored to assess their impact on snoRNA expression. Taken together, these results suggest that snoRNA expression profiles could be used as new biomarkers to refine the classification of IGHV-M CLL and should be further explored to ascertain the functional link between progression and non-coding RNA modulation. The authors thank the patients who participated in this study, Frédéric Martins and Jean-José Maoret (GeT-Purpan genomic platform, Toulouse, France). PB is supported by the Institut Universitaire de France and the LABEX TOUCAN (Laboratoire d'Excellence Toulouse Cancer). LB is supported by the Fondation ARC (Association pour la Recherche sur le Cancer). LB, WV, SG, CQ and OZ performed the experiments; LB, WV, MB, LY and PB designed the study; AQ-M, LY and FD collected and assembled the clinical records; LB, WV and MB analysed and interpreted the data; LB, MB, LY and PB prepared the first draft and finalized the manuscript; and all authors contributed to the writing of the manuscript and gave final approval. The authors declare no competing financial interests. Data S1. Supplementary methods. Fig S1. SnoRNA expression profiles in CLL and CD19+ control samples. Fig S2. A 20 snoRNA signature identifies low- and high-risk CLL among IGHV-mutated patients. Fig S3. 20 snoRNA signature expression index. Fig S4. CFSE dilution after proliferation induction in CLL and normal B-cells. Fig S5. Expression levels of the 20 snoRNAs in the signature after proliferation induction in CLL. Fig S6. Expression levels of the 20 snoRNAs in the signature after proliferation induction in normal B-cells. Table SI. Patient characteristics of the exploration and validation cohorts. Table SII. List of the primers used in the Fluidigm experiment. Table SIII. Name, targets, host gene and localization of the 20 snoRNAs from the signature. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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.

Here, it was determined that chronic lymphocytic leukemia (CLL) cells express the α subunit, but not the β subunit, of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR/CSF2R). GM-CSFRα was detected on the surface, in the cytosol, and in the nucleus of CLL cells via confocal microscopy, cell fractionation, and GM-CSFRα antibody epitope mapping. Because STAT3 is frequently activated in CLL and the GM-CSFRα promoter harbors putative STAT3 consensus binding sites, MM1 cells were transfected with truncated forms of the GM-CSFRα promoter, then stimulated with IL6 to activate STAT3 and to identify STAT3-binding sites. Chromatin immunoprecipitation (ChIP) and an electoromobility shift assay (EMSA) confirmed STAT3 occupancy to those promoter regions in both IL6-stimulated MM1 and CLL cells. Transfection of MM1 cells with STAT3-siRNA or CLL cells with STAT3-shRNA significantly downregulated GM-CSFRα mRNA and protein levels. RNA transcripts, involved in regulating cell survival pathways, and the proteins KAP1 (TRIM28) and ISG15 coimmunoprecipitated with GM-CSFRα. GM-CSFRα–bound KAP1 enhanced the transcriptional activity of STAT3, whereas GM-CSFRα-bound ISG15 inhibited the NF-κB pathway. Nevertheless, overexpression of GM-CSFRα protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFRα knockdown induced apoptosis in CLL cells, suggesting that GM-CSFRα provides a ligand-independent survival advantage. Implications: Constitutively, activation of STAT3 induces the expression of GM-CSFRα that protects CLL cells from apoptosis, suggesting that inhibition of STAT3 or GM-CSFRα may benefit patients with CLL. Mol Cancer Res; 12(9); 1267–82. ©2014 AACR.

Upon GM-CSF binding, the α and β subunits of the GM-CSF receptor (GM-CSFR) dimerize and activate signaling. Because of a potential role for GM-CSF in treating CLL, we sought to determine whether this mechanism is operative in CLL cells.

MicroRNA (miR) deregulation is a hallmark of CLL. However little is known about the mechanisms of miR gene transcription. Because in CLL signal transducer and activator of transcription (STAT)-3 is constitutively phosphorylated on serine 727 residues and activates protein-coding genes known to be induced by phosphotyrosine STAT3, we sought to determine whether phosphoserine STAT3 also activates protein non-coding genes.

MicroRNA (miR) abnormalities play a key role in the pathogenesis of chronic lymphocytic leukemia (CLL). High levels of miR-155 have been detected in human neoplasms, and overexpression of miR-155 has been found to induce lymphoma in mice. High levels of miR-155 were detected in CLL cells and STAT3, which is known to induce miR-21 and miR-181b-1 expression, is constitutively activated in CLL. Given these findings, we hypothesized that STAT3 induces miR-155. Sequence analysis revealed that the miR-155 promoter harbors two putative STAT3 binding sites. Therefore, truncated miR-155 promoter constructs and STAT3 small interfering RNA (siRNA) were co-transfected into MM1 cells. Of the two putative binding sites, STAT3-siRNA reduced the luciferase activity of the construct containing the 700-709 bp STAT3 binding site, suggesting that this site is involved in STAT3-induced transcription. Electrophoretic mobility shift assay confirmed that STAT3 bound to the miR-155 promoter in CLL cells, and chromatin immunoprecipitation and luciferase assay confirmed that STAT3 bound to the 700-709 bp but not the 615-624 bp putative STAT3 binding site in CLL cells. Finally, STAT3-small hairpin RNA downregulated miR-155 gene expression, suggesting that constitutively activated STAT3 binds to the miR-155 gene promoter. Together, these results suggest that STAT3 activates miR-155 in CLL cells.

BACKGROUND:Several methods are available to detect MRD in patients with CML in complete molecular remission (CMR) and taking tyrosine kinase inhibitor (TKI) therapy.MATERIALS AND METHODS:We performed clonogenic assays on mononuclear bone marrow cells from 14 patients. Of the 10 assessable samples, 6 were from patients in CMR and 4 from patients in complete cytogenetic remission but had detectable MRD using polymerase chain reaction (PCR) analysis (positive controls). At least 10 colonies per sample were microaspirated and individual colonies were subjected to PCR analysis.RESULTS:Of the 6 patients in CMR, 5 harbored breakpoint cluster region abelson (BCR-ABL1) negative colonies but in 1 sample, 1 of the 10 colonies analyzed was positive for BCR-ABL1. Of the 4 patients with evidence of MRD in peripheral blood, 2 had negative and 2 had positive BCR-ABL1 colonies.CONCLUSION:MRD is still detectable using clonogenic assays in some patients with CML after achieving CMR using TKI therapy, which is likely responsible for relapse on TKI discontinuation. Because of the large number of single colonies that need to be analyzed, the use of clonogenic assays in clinical practice to determine the feasibility of TKI discontinuation is not recommended.

In addition to the canonical right-handed double helix, DNA molecule can adopt several other non-B DNA structures. Readily formed in the genome at specific DNA repetitive sequences, these secondary conformations present a distinctive challenge for progression of DNA replication forks. Impeding normal DNA synthesis, cruciforms, hairpins, H DNA, Z DNA and G4 DNA considerably impact the genome stability and in some instances play a causal role in disease development. Along with previously discovered dedicated DNA helicases, the specialized DNA polymerases emerge as major actors performing DNA synthesis through these distorted impediments. In their new role, they are facilitating DNA synthesis on replication stalling sites formed by non-B DNA structures and thereby helping the completion of DNA replication, a process otherwise crucial for preserving genome integrity and concluding normal cell division. This review summarizes the evidence gathered describing the function of specialized DNA polymerases in replicating DNA through non-B DNA structures.

Abstract 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.

BACKGROUNDS:Approximately 1,000 microRNAs (miRs) are present in the human genome; however, little is known about the regulation of miR transcription. Because miR levels are deregulated in chronic lymphocytic leukemia (CLL) and signal transducer and activator of transcription (STAT)-3 is constitutively activated in CLL, we sought to determine whether STAT3 affects the transcription of miR genes in CLL cells.METHODS:We used publically available data from the ENCODE project to identify putative STAT3 binding sites in the promoters of miR genes. Then we transfected CLL cells with STAT3-shRNA or with an empty vector, and to determine which miRs are differentially expressed, we used a miR microarray approach followed by validation of the microarray results for 6 miRs using quantitative real-time polymerase chain reaction (qRT-PCR).RESULTS:We identified putative STAT3 binding sites in 160 promoter regions of 200 miRs, including miR-21, miR-29, and miR-155, whose levels have been reported to be upregulated in CLL. Levels of 72 miRs were downregulated (n = 63) or upregulated (n = 9). qRT-PCR confirmed the array data in 5 of 6 miRs.CONCLUSIONS:The presence of activated STAT3 has a profound effect on miR expression in CLL cells.

Abstract 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.

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 (r p = 0.28, P P = 0.01), suggesting that previous exposure to chemotherapy activated compensatory survival pathways. Conclusions: Spontaneous apoptosis occurs in PB CLL cells of all patients regardless of disease stage or cytogenetic abnormality. High PB lymphocyte count linearly correlates with increased spontaneous apoptosis rates and decreased intracellular STAT3 levels. Disclosures: No relevant conflicts of interest to declare.

Abstract 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.