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Highly expressed urokinase plasminogen activator receptor (uPAR) can interact with ␣5␤1 integrin leading to persistent ERK activation and tumorigenicity. Disrupting this interaction reduces ERK activity, forcing cancer cells into dormancy. We identified a site in uPAR domain III that is indispensable for these effects. A 9-mer peptide derived from a sequence in domain III (residues 240 -248) binds purified ␣5␤1 integrin. Substituting a single amino acid (S245A) in this peptide, or in full-length soluble uPAR, impairs binding of the purified integrin. In the recently solved crystal structure of uPAR the Ser-245 is confined to the large external surface of the receptor, a location that is well separated from the central urokinase plasminogen binding cavity. The impact of this site on ␣5␤1 integrin-dependent cell functions was examined by comparing cells induced to express uPAR wt or the uPAR S245A mutant. Transfecting uPAR wt into cells with low endogenous levels of uPAR, inactive integrin, low ERK activity, and a dormant phenotype in vivo restores these functions and reinstates growth in vivo. In contrast, transfection of the same cells with uPAR S245A elicits only very small changes. Incubation of highly malignant cells with the wild-type, but not the S245A mutant peptide, disrupts the uPAR integrin interaction leading to down-regulation of ERK activity. The relevance of this binding site, and of the lateral uPAR-␣5␤1 integrin interaction, to ERK pathway activation and tumor growth implicates it as a possible specific target for cancer therapy.

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The natural killer (NK) type of lymphoproliferative disease of granular lymphocytes (LDGL) is associated with the expansion of CD3 ؊ , CD16 ؉ , and/or CD56 ؉ lymphocytes. We have examined the repertoire of NK receptors expressed on these cells and delineated the functional activity. We found skewed NK receptor expression on patient NK cells. Reactivity to a single anti-killer cell immunoglobulinlike receptor (anti-KIR) antibody was noted in 7 of 13 patients. LDGL patients variably expressed NKp30, NKp44, and NKp46 RNA. In contrast, CD94 and its inhibitory heterodimerization partner NKG2A were homogenously expressed at high levels on these NK cells. Interestingly, these patients expressed a large number of activating KIR receptors by genotype analysis. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that lower than normal levels of RNA of the inhibitory KIR was present in some patients in contrast to normal NK cells. Consistent with a high level of activating receptors, we found the NK-LDGL cells have potent cytolytic function in both direct and redirected cytotoxicity assays. These results demonstrate that patients with NK-LDGL have an increased activating-to-inhibitory KIR ratio. This altered ratio might induce inappropriate lysis or cytokine production and impact the disease pathogenesis. ( IntroductionThe natural killer (NK) type of lymphoproliferative disease of granular lymphocytes (LDGL) is characterized by the expansion of NK cells that express CD56 and/or CD16 but do not express CD3 or the T-cell receptor (TCR) complex or rearrange TCR genes. [1][2][3][4] Most cases of LDGL in the United States and Europe follow a chronic clinical course. 1,5 Aggressive cases of NK leukemia are more commonly seen in the Far East and are associated with a rapidly progressive clinical course with a median survival of only months. Epstein Barr virus (EBV) has been reported as an etiologic agent of aggressive NK leukemia/lymphoma. 6,7 The etiology of LDGL is not known. However, we have found that sera from 67% of LDGL patients have high titer-specific antibody reactivity directed at a 34-amino acid epitope of the envelope protein of human T-cell lymphotropic virus type I (HTLV-I). 8 This finding raises the possibility of serologic crossreactivity to a cellular or retroviral antigen showing some amino acid homology with the transmembrane glycoprotein of HTLV. However, these patients are not infected with any of the known prototypic retroviruses such as HTLV-I, HTLV-II, primate Tlymphotropic virus (PTLV), HIV, or bovine leukemia virus (BLV). 9,10 We hypothesize that chronic viral stimulation by a novel HTLV-related retrovirus with homology in the p21e region may contribute to the pathogenesis of this disease.Multiple NK-associated receptors have now been identified, which might prove useful in the clinical diagnosis of LDGL as well as provide a better understanding of disease pathogenesis. Normal NK cells express 3 types of receptors: C-type lectin receptors (NKG2), immunoglobulin-like r...

In prostate cancer (PCa), the functional synergy between androgen receptor (AR) and nuclear factor-κ B (NF-κB) escalates the resistance to therapeutic regimens and promotes aggressive tumor growth. Although the underlying mechanisms are less clear, gene regulatory abilities of coactivators can bridge the transcription functions of AR and NF-κB. The present study shows that MYST1 (MOZ, YBF2 and SAS2, and TIP60 protein 1) costimulates AR and NF-κB functions in PCa cells. We demonstrate that activation of NF-κB promotes deacetylation of MYST1 by sirtuin 1. Further, the mutually exclusive interactions of MYST1 with sirtuin 1 vs AR regulate the acetylation of lysine 16 on histone H4. Notably, in AR-lacking PC3 cells and in AR-depleted LNCaP cells, diminution of MYST1 activates the cleavage of poly(ADP-ribose) polymerase and caspase 3 that leads to apoptosis. In contrast, in AR-transformed PC3 cells (PC3-AR), depletion of MYST1 induces cyclin-dependent kinase (CDK) N1A/p21, which results in G2M arrest. Concomitantly, the levels of phospho-retinoblastoma, E2F1, CDK4, and CDK6 are reduced. Finally, the expression of tumor protein D52 (TPD52) was unequivocally affected in PC3, PC3-AR, and LNCaP cells. Taken together, the results of this study reveal that the functional interactions of MYST1 with AR and NF-κB are critical for PCa progression.

IntroductionCord blood (CB) cells are an effective source of pluripotent hematopoietic stem and progenitor cells (HSCs/HPCs) that can be used to hematologically reconstitute patients who have received myeloablative chemotherapy and/or radiation therapy. 1 Numerous investigators have tried to increase CB stem cell numbers by culturing CD34 ϩ cells ex vivo under a variety of culture conditions; these attempts have, however, met with limited success. 2-13 CBHSCs/HPCs have also been used to generate alternative sources of terminally differentiated blood cells for use as transfusion products. 4,8 Adult blood HSCs/HPCs, human embryonic stem cells (ES) as well as induced pluripotent stem cells (iPS) have also been used for this same purpose. 4,8,14,15 All presently available TPs are composed of terminally differentiated cells with a finite life span. 2,4,8 HPCs retain their capacity to undergo repeated divisions allowing them to produce greater numbers of blood cells belonging to a particular lineage. The use of HPCs as a TP would represent a major paradigm shift because they would continue to generate differentiated cells for a prolonged period of time. The major obstacle to the creation of such a TP is the lack of sufficient numbers of HSCs/HPCs from a readily accessible sources. [16][17][18] Recently, a variety of epigenetic events including the methylation of gene promoter regions as well as transcriptional inhibitory complexes such as histone deacetylases and certain histone methylases have been shown to influence HSC fate decisions. 19,20 Chromatin-modifying agents (CMAs) such as HDACIs and DNA methyltransferase inhibitors (DNMTIs) comprise a structurally diverse group of compounds which are capable of regulating chromatin remodeling and influencing gene expression patterns. 21,22 Our laboratory has sequentially treated both adult marrow and CB-CD34 ϩ cells with a DNMTI followed by an HDACI in vitro resulting in an increase in the numbers of human marrow repopulating cells (MRCs). 23,24 The use of such small molecules to alter normal HSC/HPC fate decisions has been confirmed by several additional laboratories. 13,19,[25][26][27] We hypothesize that a particular combination of cytokines and an HDACI might be useful to promote erythroid commitment of CB-CD34 ϩ cells. Methods Isolation of CB-CD34 ؉ cellsFresh CB collections were obtained from the Placental Blood Program at the New York Blood Center (New York, NY) according to guidelines established by the Mount Sinai School of Medicine Institutional Review Board. Low-density CB-mononuclear cells (MNCs) were isolated by density centrifugation and CD34 ϩ cells were isolated as previously described. 23 Highly purified CB-CD34 ϩ cells (90%-98%) were used for all experiments. Ex vivo cultureHuman CB-CD34 ϩ cells (1 ϫ 10 5 /mL) were cultured in Iscove modified Dulbecco medium (IMDM; Lonza) containing 30% FBS (HyClone Laboratories) supplemented with 100 ng/mL SCF, 100 ng/mL Flt3 ligand (FL), 100 ng/mL Tpo, and 50 ng/mL IL-3 (cytokines were a gift of Amgen) and incubated in...

The myeloproliferative neoplasms (MPNs) are a group of clonal hematological malignancies characterized by a hypercellular bone marrow and a tendency to develop thrombotic complications and to evolve to myelofibrosis and acute leukemia. Unlike chronic myelogenous leukemia, where a single disease-initiating genetic event has been identified, a more complicated series of genetic mutations appear to be responsible for the BCR-ABL1-negative MPNs which include polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Recent studies have revealed a number of epigenetic alterations that also likely contribute to disease pathogenesis and determine clinical outcome. Increasing evidence indicates that alterations in DNA methylation, histone modification, and microRNA expression patterns can collectively influence gene expression and potentially contribute to MPN pathogenesis. Examples include mutations in genes encoding proteins that modify chromatin structure (EZH2, ASXL1, IDH1/2, JAK2V617F, and IKZF1) as well as epigenetic modification of genes critical for cell proliferation and survival (suppressors of cytokine signaling, polycythemia rubra vera-1, CXC chemokine receptor 4, and histone deacetylase (HDAC)). These epigenetic lesions serve as novel targets for experimental therapeutic interventions. Clinical trials are currently underway evaluating HDAC inhibitors and DNA methyltransferase inhibitors for the treatment of patients with MPNs.

340 Presently, blood transfusion products (TP) are composed of terminally differentiated cells with a finite life span. We attempted to develop an alternative TP which would be capable of generating additional red blood cells (RBC). Several histone deacetylase inhibitors (HDACIs) were used in vitro to reprogram cord blood (CB) CD34+ cells to differentiate to erythroid progenitor cells (EPC). We demonstrated that CB CD34+ cells in the presence of HDACIs (SAHA, VPA and TSA), and a combination of cytokines SCF, IL-3, TPO and FLT3, promoted expansion of CD34+ cells and CD34+CD90+ cells as compared to cultures containing cytokines alone. Addition of VPA resulted in the greatest expansion of CD34+ cells, CD34+CD90+cells+ (59.4 fold, p=0.01; 66.7 fold, p=0.02, respectively) as compared to SAHA and TSA. VPA also led to the generation of the greatest absolute number of EPC cells (14.9×106, p=0.002), approximately a 5500 fold in the numbers of assayable EPC, as compared to primary CB. The single cell analyses of CB CD34+ cells (Day0) and single CD34+ reisolated from ex-vivo cultures pretreated with cytokines alone or cytokines+VPA demonstrated an skewed differentiation program of CD34+ cells to EPC (>94%, p=0.003) compared to CB CD34+(50%) and cytokines alone (29%). We investigated the expression of lineage specific phenotypic markers expressed by CD34+ cells exposed to cytokines alone or cytokines plus VPA. The FACS analyses showed a significantly greater proportion of CD34+CD36+ (52.4% vs 21.0%) CD36+CD71+(44.5% vs7.6%), CD36+GPA+(12.8% Vs 4.0%) and CD71+GPA+(22.2% vs 6.3%) cells with lower numbers of CD19+(2.8% vs 13.6%) cells, CD14+(2.0% vs 8.9%), CD15+(1.8 vs 6.9%) in VPA treated CD34+ cells as compared to cytokines alone. We monitored the relative expression of a group of genes characteristic of both primitive HPC and erythroid commitment (Bmi1, Dnmt1, Ezh2, Smad5, Eklf, GATA1, GATA2, EpoR and Pu.1). Q-PCR was performed on CD34+cells reisolated from cultures treated with cytokines alone or cytokines plus VPA and compared to primary CB CD34+ cells. The expression of genes associated with retention of the biological properties of the primitive HPC (Bmi1-2.6 fold, Dnmt1-10.3 fold and Ezh2-4.8 fold) and erythroid lineage specific genes (Smad5-6.2 fold, GATA2-3.7 fold) were upregulated and Pu.1 (0.6-fold), GATA1(1.9 fold) were downregulated as compared to cytokines alone. However, expression of EpoR and Eklf were similar in the two cell populations Histone acetylation study showed that the CB CD34+ cells and VPA treated CD34+ cells had a significant proportion of acetylated H3K9 cells, 52.2% and 56.1% respectively, while this population was virtually absent in CD34+ cells exposed to cytokines alone (1.3%, p=0.001). ChIP assay demonstrated a varying degree of H3K9/14 and H3K27 acetylation within the promoters of VPA treated CD34+ cells for GATA2 (7.4 fold, 7.2 fold), Eklf (7.4 fold, 9.7 fold), Pu.1(4.5fold, 4.8 fold), EpoR (2.3 fold, 4.7 fold) and GATA1(4.7 fold, 2.9 fold). The acetylation of cytokines treated CD34+ cells were much lower than VPA treated CD34+ cells. The VPA treated cell product after 9 days (supplemented with SCF, Epo and IL-3 for 2 additional days) compared to 7 days contained a greater percentage of EPC and erythroid precursor cells CD34+CD36+(24.9% vs 23.0%), CD36+GPA+(33.9% vs 18.8%), CD36+. CD71+(55.8% vs 37.8%), CD71+GPA+(33.9% vs 20.5%) and CD34+CXCR4+(28.8% vs 21.0 %). The TP contained very limited number of CD19+(1.4%), CD14+(11.11%) or CD15+(6.8%) of cells. Approximately 50 % of the cells present in the TP expressed the chemokine receptor CXCR4. We next evaluated the behavior of ex vivo expanded cell product following transfusion into sublethally irradiated NOD/SCID mice. FACS analyses of mice peripheral blood (PB) on serial days showed evidence of circulating nucleated erythroid and enucleated red cells. The greatest number of circulating human RBC (12.4%±6.8%) was observed on day5. RT-PCR analyses on the PB of mice on day 15 revealed the presence of erythroid cells containing both human adult and fetal hemoglobin. On day 15 the mice were sacrificed and the degree of human cells engraftment in the marrow were predominately hu -CD45+ (7.4%), CD34-CD36+(1.8%), CD36 (4.5%) and GPA+(1.7%) with no evidence of CD33+, CD14+, CD19+ and CD41+ cells. The ex vivo generated EPC-TP likely represents a paradigm shift in transfusion medicine due to its continued ability to generate additional RBC. Disclosures: No relevant conflicts of interest to declare.