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Supplementary Figure 1 from Nm23-H1 Indirectly Promotes the Survival of Acute Myeloid Leukemia Blast Cells by Binding to More Mature Components of the Leukemic Clone

In light of the chemical exploitation of CO2, new reusable materials for efficiently catalyzing the cycloaddition of CO2 and epoxides under moderate conditions are needed. Herein, a new series of i...

In light of the chemical exploitation of CO

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)(2)D3,] possess in vitro multiple anti-cancer activities including growth arrest, induction of apoptosis and differentiation of a variety of different types of malignant cells. However, its use as a therapeutic agent is hindered by its calcemic effects. Analogs of 1,25(OH)(2)D3 have enhanced anti-tumor activity, with reduced calcemic effects. However, limited clinical studies using vitamin D compounds have not yet achieved major clinical success. Nevertheless, pre-clinical studies suggest that the combination of either 1,25(OH)(2)D3 or its analogs with other agents can have additive or synergistic anti-cancer activities, suggesting future clinical studies.

Human sex determining region Y-box 2 (SOX2) is an important transcriptional factor involved in the pluripotency and stemness of human embryonic stem cells. SOX2 plays important roles in maintaining cancer stem cell activities of melanoma and cancers of the brain, prostate, breast, and lung. SOX2 is also a lineage survival oncogene for squamous cell carcinoma of the lung and esophagus. Spontaneous cellular and humoral immune responses against SOX2 present in cancer patients classify it as a tumor-associated antigen (TAA) shared by lung cancer, glioblastoma, and prostate cancer among others. In this study, B-cell epitopes were predicted using computer-assisted algorithms. Synthetic peptides based on the prediction were screened for recognition by serum samples from cancer patients using ELISA. Two dominant B-cell epitopes, SOX2:52-87 and SOX2:98-124 were identified. Prostate cancer, glioblastoma and lung cancer serum samples that recognized the above SOX2 epitopes also recognized the full-length protein based on Western blot. These B-cell epitopes may be used in assessing humoral immune responses against SOX2 in cancer immunotherapy and stem cell-related transplantation.

NY-ESO-1 is a cancer/germline antigen (Ag) with distinctively strong immunogenicity. We have previously demonstrated that NY-ESO-1 serves as an endogenous adjuvant by engaging dendritic cell (DC)-surface receptors of calreticulin (CRT) and toll-like receptor (TLR) 4. In the present study, NY-ESO-1 was investigated for its immunomodulatory roles as a molecular adjuvant in whole-tumor cell vaccines using the Renca kidney cancer model. Renca cells were genetically engineered to express NY-ESO-1 on the cell surface to enhance direct interactions with DC. The effect of ectopic cell-surface expression of NY-ESO-1 was investigated on tumor immunogenicity, DC activation, cytotoxic T lymphocytes against model tumor-associated Ags, and the effectiveness of the modified tumor cells as a therapeutic whole-cell vaccine. Cell-surface expression of NY-ESO-1 was able to reduce the tumor growth of Renca cells in BALB/c mice, although the modification did not alter cell proliferation rate in vitro. Directly engaging the innate immune system through NY-ESO-1 facilitated the interaction of tumor cells with DC, leading to enhanced DC activation and subsequent tumor-specific T-cell priming. When used as a therapeutic whole-cell vaccine, Renca cells with NY-ESO-1 on the surface mediated stronger inhibitory effects on tumor growth and metastasis compared with parental Renca or Renca cells expressing a control protein GFP on the surface. Augmented antitumor efficacy correlated with increased CD8 T-cell infiltration into tumors and decreased myeloid-derived suppressor cells and regulatory T cells in the spleen. As a cancer/germline Ag and as an immunomodulatory adjuvant through engaging innate immune receptors, NY-ESO-1 offers a unique opportunity for improved whole-tumor cell vaccinations upon the classic GM-CSF-engineered cell vaccines.

Modeling human body response to dynamic loading events and developing biofidelic human surrogate systems require accurate material properties over a range of loading rates for various human organ tissues. This work describes a technique for measuring the shear properties of soft biomaterials at high rates of strain (100–1000 s −1 ) using a modified split Hopkinson pressure bar (SHPB). Establishing a uniform state of stress in the sample is a fundamental requirement for this type of high-rate testing. Input pulse shaping was utilized to tailor and control the ramping of the incident loading pulse such that a uniform stress state could be maintained within the specimen from the start of the test. Direct experimental verification of the stress uniformity in the sample was obtained via comparison of the force measured by piezoelectric quartz force gages on both the input and the output sides of the shear specimen. The technique was demonstrated for shear loading of silicone gel biosimulant materials and porcine brain tissue. Finite element simulations were utilized to further investigate the effect of pulse shaping on the loading rate and rise time. Simulations also provided a means for visualization of the degree of shear stress and strain uniformity in the specimen during an experiment. The presented technique can be applied to verify stress uniformity and ensure high quality data when measuring the dynamic shear modulus of soft biological simulants and tissue.

Nm23-H1 plays complex roles in the development of diverse cancers including breast carcinoma, high-grade lymphomas, and acute myeloid leukemia (AML). In the case of AML and lymphomas, serum Nm23-H1 protein is elevated with the highest levels correlating with poorest prognosis. A recent study identified that this association is most likely causal in AML and that Nm23-H1 acts as an AML cell survival factor. In this study, we report heterogeneity in the ability of AML samples to bind and respond to Nm23-H1, and we offer evidence that binding is essential for improved survival. Further, we show that the subset of AMLs that bind Nm23-H1 do not do so through the putative Nm23-H1 receptor MUC1*. Although rNm23-H1 promoted the survival of the most primitive blasts within responding AMLs, it was not these cells that actually bound the protein. Instead, rNm23-H1 bound to more mature CD34lo/CD34− and CD11b+ cells, revealing an indirect survival benefit of Nm23-H1 on primitive blasts. In support of this finding, the survival of purified blast cells was enhanced by medium conditioned by more mature cells from the clone that had been stimulated by rNm23-H1. Levels of interleukin 1β (IL1β) and IL6 in rNm23-H1 conditioned medium mirrored the potency of the conditioned media to promote blast cell survival. Furthermore, Nm23-H1 expression was significantly associated with IL1β and IL6 expression in primary uncultured AML samples. These findings have implications for the role of Nm23-H1 in AML and its use as a prognostic marker. Additionally, they offer the first evidence of novel cross-talk between cell populations within the tumor clone. Cancer Res; 71(3); 1177–86. ©2010 AACR.

Current experimental models of blast injuries used to study blast-induced neurotrauma (BINT) vary widely, which makes the comparison of the experimental results extremely challenging. Most of the blast injury models replicate the ideal Friedländer type of blast wave, without the capability to generate blast signatures with multiple shock fronts and refraction waves as seen in real-life conditions; this significantly reduces their clinical and military relevance. Here, we describe the pathophysiological consequences of graded blast injuries and BINT generated by a newly developed, highly controlled, and reproducible model using a modular, multi-chamber shock tube capable of tailoring pressure wave signatures and reproducing complex shock wave signatures seen in theater. While functional deficits due to blast exposure represent the principal health problem for today's warfighters, the majority of available blast models induces tissue destruction rather than mimic functional deficits. Thus, the main goal of our model is to reliably reproduce long-term neurological impairments caused by blast. Physiological parameters, functional (motor, cognitive, and behavioral) outcomes, and underlying molecular mechanisms involved in inflammation measured in the brain over the 30 day post-blast period showed this model is capable of reproducing major neurological changes of clinical BINT.

Identification of marker genes including RUNX3 ( AML2 ) that discriminate between different myeloproliferative neoplasms and normal individuals

Purpose: The histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), has multiple antitumor effects against a variety of human cancers. Experimental design: We treated several anaplastic and papillary thyroid cancer cell lines with SAHA to determine if it could inhibit the growth of these cells in vitro and in vivo. Results: SAHA effectively inhibited 50% clonal growth of the anaplastic thyroid cancer cell lines, ARO and FRO, and the papillary thyroid cancer cell line, BHP 7-13, at 1.3 × 10−7 to 5 × 10−7 mol/L, doses that are achievable in patients. In concert with growth inhibition, SAHA down-regulated the expression of cyclin D1 and up-regulated levels of p21WAF1. Annexin V and cleavage of poly(ADP)ribose polymerase were both increased by exposure of the thyroid cancer cells to SAHA. Expression of the death receptor 5 (DR5) gene was also increased by SAHA, but the combination of the DR5 ligand, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), with SAHA had little effect compared with SAHA alone. Of note, the combination of paclitaxel, doxorubicin, or paraplatin with SAHA enhanced cell killing of the thyroid cancer cells. In addition, murine studies showed that SAHA administered daily by i.p. injection at 100 mg/kg inhibited the growth of human thyroid tumor cells. Conclusion: Our data indicate that SAHA is a plausible adjuvant therapy for thyroid cancers.

Vitamin D3 is produced in skin and is sequentially metabolized by the liver and kidney to the biologically active form 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. It is a seco-steroid hormone that regulates calcium homeostasis within the body. The genomic actions of 1,25(OH)2D3 are modulated through the vitamin D receptor (VDR). VDR belongs to a superfamily of nuclear receptors that transduce hormonal signals from the immediate environment and transactivate genes in response to these signals. Target genes contain hormone response elements (VDREs) in their promoters to which heterodimers of VDR and retinoid X receptors (RXR) can bind and transactivate expression of the target genes. The VDR is expressed in at least 30 different target tissues including bone, kidney, blood, breast, prostate, gut, activated B- and T- lymphocytes, monocytes and keratinocytes). Most dividing cell types, normal and malignant, can express VDR and respond to 1,25(OH)2D3. Although 1,25(OH)2D3 and its analogs (termed deltanoids) are important regulators of calcium and bone metabolism, their non-calciotropic activities that include inhibition of cell proliferation, promotion of cell differentiation and modulation of immune cell function have spurred interest in therapeutic applications in a wide variety of diseases. In this report, the anticancer and newly discovered antimicrobial actions of 1,25(OH)2D3 and deltanoids are reviewed.

We and others have demonstrated expression of the aldo-keto reductase AKR1C3 in myeloid leukemia cell lines and that inhibitors of the enzyme, including nonsteroidal anti-inflammatory drugs (NSAIDs), promote HL-60 differentiation in response to all-trans retinoic acid (ATRA) and 1alpha,25-dihydroxyvitamin D3 (D3). Here, we demonstrate that overexpression of AKR1C3 reciprocally desensitizes HL-60 cells to ATRA and D3, thus confirming the enzyme as a novel regulator of cell differentiation. AKR1C3 possesses marked 11-ketoreductase activity converting prostaglandin (PG) D2 to PGF2alpha. Supplementing HL-60 cultures with PGD2 mimicked treatment with AKR1C3-inhibitors by enhancing the differentiation of the cells in response to ATRA. However, PGD2 is chemically unstable, being converted first to PGJ2 and then stepwise to 15-deoxy-Delta(12,14)-prostaglandin J2(15Delta-PGJ2), a natural ligand for the peroxisome proliferator-activated receptor-gamma (PPARgamma). Consistent with this, PGD2 was rapidly converted to PGJ2 under normal tissue culture conditions but not in the presence of recombinant AKR1C3 when PGF2alpha was predominantly formed. In addition, PGJ2 but not PGF2alpha recapitulated the potentiation of HL-60 differentiation by PGD2 and AKR1C3 inhibitors. Furthermore, the capacity of all of these treatments to potentiate HL-60 cell differentiation was significantly reduced in the presence of the PPARgamma-antagonist GW 9662. We conclude that AKRIC3 protects HL-60 cells against ATRA and D3-induced cell differentiation by limiting the production of natural PPARgamma ligands via the diversion of PGD2 toward PGF2alpha and away from PGJ2. In addition, these observations identify AKR1C3 as plausible target for the non-cyclooxygenase-dependent antineoplastic actions of NSAIDs.

Abstract 2968 Poster Board II-944 Previously, we identified and validated PIM1 as a differentially expressed gene in mantle cell lymphoma (MCL) patient samples. Further, we have shown PIM1 to be a significant prognostic biomarker in MCL. PIM1 is a serine/threonine kinase that is transcriptionally regulated by cytokines, mitogens, and numerous growth factors. PIM1 cooperates with other oncogenes in tumorigenesis and has been implicated in the development of leukemias, lymphomas, late progression events, and most recently in prostate cancer. PIM1 is overexpressed in aggressive lymphomas, such as the blastoid variant of MCL, and the ABC-subtype of diffuse large B-cell lymphomas (DLBCL). Here, we tested the in vivo cooperation of PIM1 with TCL1 in murine lymphomagenesis by producing double transgenic murine strains. PIM1 transgenic mice overexpress murine PIM1 under the control of the immunoglobulin enhancer Eμ. TCL1 transgenic mice (pEμ-B29-TCL1) fail to down-regulate TCL1 expression in mature B and T cells and provide a unique model for mature B-cell malignancies. We hypothesized that PIM1 would either accelerate TCL1-driven lymphomagenesis, result in the development of immature lymphomas, or both. Lymphoid malignancies were examined by immunohistochemistry and classified by a hematopathologist according to “Mouse Models of Human Blood Cancers” (Li et al., 2008). A Kaplan-Meier plot demonstrated statistically significant acceleration of lymphomagenesis in the PIM1/TCL1 transgenic mice when compared with the single transgenic strains. The median lymphoma-free survival for TCL1 single transgenic mice, PIM1 single transgenic mice, or PIM1/TCL double transgenic mice were 10.0 months, 16.0 months, and 8.5 months, respectively. The results were statistically significant: TCL1 vs. PIM1/TCL1 (p=0.0008), PIM1 vs. PIM1/TCL1 (p12 months of age) B-cell lymphomas. The most common lymphomas in PIM1 single transgenic were low-grade B-cell lymphomas [12 of 31 (38.7%)], mainly follicular lymphomas. A minority of PIM1 single transgenic mice developed aggressive lymphomas [6 of 31 (19.4%)], including DLBCL and Burkitt's lymphoma. In contrast, the majority of TCL1 transgenic mice developed aggressive B-cell lymphomas [21 of 36 (58.3%)], mainly DLBCL, lymphohistiocytic subtype. The majority of PIM1/TCL1 double transgenic mice also developed aggressive B-cell lymphomas [20 of 34 (58.8%)], mainly DLBCL, lymphohistiocytic subtype. The low-grade lymphomas that developed in PIM1/TCL1 mice included 5 cases of lymphoplasmacytic lymphoma (LPL); one of these cases had transformed in addition to a DLBCL. Further, endogenous expression of PIM kinase family members was investigated in a human lymphoma cell line bank (n=40) by quantitative real-time PCR. PIM1, PIM2, and PIM3 were found to be overexpressed in cell lines derived from human lymphoid malignancies of multiple histologies. In summary, aberrant PIM1 overexpression in TCL1 transgenic mice accelerated the development of mature, aggressive B-cell lymphomas. The classification of lymphomas in PIM1/TCL1 mice revealed similar histologies as in TCL1 single transgenic mice, mainly DLBCL. Single transgenic PIM1 mice developed low-grade B-cell lymphomas after prolonged observation time. The expression of all 3 PIM kinase family members in human lymphomas implies that pan-PIM kinase inhibitors should be developed as a potential mechanism of drug resistance to more restricted PIM inhibitors could be compensatory overexpression of the non-targeted Pim family members. A clinical trial with a pan-PIM inhibitor is currently ongoing. Disclosures: No relevant conflicts of interest to declare.

Despite the success of combination chemotherapy for the treatment of Hodgkin lymphoma (HL), chemotherapy-resistant disease still remains an unresolved problem with most patients eventually dying due to progression. HL is an ideal disease for targeted therapy. CD30 is a 120-kDa transmembrane glycoprotein belonging to the tumor necrosis factor (TNF)-receptor superfamily and strongly expressed in HL Reed-Sternberg cells. We hypothesize that specific, antibody-mediated, CD30-directed delivery of lentiviral vector constructs, encoding for a lethal cellular toxin, will be an effective anti-lymphoma strategy. However, gene delivery based therapy is limited by the transduction of non-target cells. The technology developed by Morizono et al. [Cell Cycle2005: 4: p854; Nature Medicine2005: 11: p346] demonstrates that lentiviruses can be specifically and effectively directed to target cells by conjugation of an antibody to a modified ZZ SINDBIS viral envelope (m168). In this study, we have used HL cell lines (CD30+: L591, L428, Hs445, RPMI6666) and Burkitt lymphoma (BL) cell lines (CD20+/CD30−: Raji, Ramos). We show that conjugation of an anti-CD30 antibody to m168 (m168anti-CD30), permits specific targeting and transduction of CD30+ Hodgkin lymphoma cells while avoiding CD30− Raji and Ramos cells [range 11–83% for HL cells versus 1–4% for BL cells]. Similarly, targeting of CD20+ cells can be achieved. Several of our HL cell lines (L591, Hs445 and RPMI6666) are CD30+/CD20+ [range 11–25% by flow cytometry] and we show that we can equally transduce these cells with an anti-CD20 antibody conjugated to our lentivirus [range 11–30% for CD20+ HL cells; 3% for L428 cells; 40–47% for BL cells]. In addition, we show in L591 cells, that the re-targeted viruses can transduce a greater percentage of target cells than an unmodified virus [83% for m168anti-CD30 versus 43% for VSVG]. These results demonstrate that the efficacy and specificity of targeted therapy can be greatly enhanced and lay the foundation for the development of more stable anti-CD30 directed lentiviral constructs expressing cellular toxins.

Quantum dots (QDs) are nanometer scale fluorescent semiconductors that have recently garnered much interest as agents for imaging. QDs have several advantages over conventional fluorescent imaging probes, including high quantum yield, broad absorption spectra and minimal photobleaching. QDs have been used little in hematology. We studied hematologic cells to determine whether they could be labeled with QDs, whether QDs could be seen after multiple cell divisions, whether QDs remained visible as cells differentiate and whether QDs could target specific antigens on the surface of cells. Using QDs attached to an endocytosed protein, we could intracellularly label all hematologic cells tested, including multiple leukemic cell lines (HL-60 and KG-1), normal human and murine bone marrow, leukemic human bone marrow (AML and CML) and normal human CD34+ cells. The QDs are easily detected by FACS and microscopy. By counting cell divisions using the cell membrane dye PKH26, we show that QDs can be seen in leukemic cell lines and primary cells through more than four cell divisions. Some cells from leukemic cell lines retain QDs for up to two weeks. We are also able to see QDs in cells through differentiation. We labeled HL-60 cells and cultured them with either 1,25 dihydroxy vitamin D or DMSO. QDs were seen in monocytic-like and neutrophil-like progeny of the labeled HL-60 cells. After labeling CD34+ cells and culturing them in appropriate cytokines, QDs were observed in the resultant monocytes. We were also able to target QDs specifically to cells expressing certain cell surface antigens. QDs complexed to streptavidin and a biotinylated anti-CD33 antibody homed to myelogenous leukemic cell lines but not lymphomatous cell lines. In summary, we show for the first time that QDs effectively label and track hematolgic cells, adding a new option when addressing cell fate questions in hematology.

Myeloproliferative disorders (MPD) are clonal stem cell diseases, which are defined by excessive production of cells in one or more hematopoietic lineages. The molecular mechanisms underlying the development of agnogenic myeloid metaplasia (AMM), polycythemia vera (PV), and essential thrombocythemia (ET) are currently poorly understood. We performed microarray analysis on 26 granulocyte samples from AMM (4), ET (5), PV (6) and normal (11) individuals in order to identify genes that: 1) distinguish MPD from normal samples and 2) distinguish between these diseases. Our data revealed a group of genes that were differentially expressed in MPD compared to normal or were differentially expressed between the three different diseases. Cytokine signaling has often been reported in development or progression of these diseases. Several of these aberrantly expressed genes included those involved in TGF-beta signaling. RUNX3 (AML2), a transcription factor that is involved in the signaling cascade mediated by TGF-beta, was markedly overexpressed in MPD (AMM 4.9-fold; PV 8.1-fold; ET 9.5-fold) compared to normal. TIEG1 (TGF-beta-inducible early growth response 1) was upregulated in AMM (6.4-fold), PV (9.5-fold), ET (16.7-fold) compared to normal. Moreover, TNFAIP3 (TNF alpha-induced protein 3) was overexpressed in MPD (AMM 13.8-fold; PV 11.6-fold; ET 9.3-fold) compared to normal, which might also suggest a potential role of TNF-alpha signaling in the pathogenesis of MPD. We also found several genes that could discriminate each disease from each other. For example, ZNF292 (zinc finger protein 292) was overexpressed in PV and ET (7.1-fold and 2.9-fold, respectively), but AMM had similar expression levels to normals. CCNL2 (cyclin L2) was overexpressed in ET (2.4-fold), unchanged in PV and downregulated by 5-fold in AMM compared to normal. Expression levels of all of these genes were confirmed by real-time PCR, and immunohistochemistry staining of normal and MPD samples for RUNX3 was comparable to our array data. We hypothesize that RUNX3 might play a role in myelopoiesis. RUNX3 mRNA levels in HL-60 cells cultured with ATRA (100 and 1000 nM) markedly (16 to 20-fold) and maximally increased by day 3. Levels subsided to control levels by day 7, suggesting that RUNX3 may be initially involved in HL-60 differentiation but returns to normal levels as these cells matured or underwent terminal differentiation. In MPD, the high levels of RUNX3 in the aberrant neutrophils might indicate that these cells are blocked at approximately the equivalent timepoint (day 3 for HL-60). Their differentiation program has been initiated, but they cannot undergo the final stages of terminal maturation as reflected by their high RUNX3 levels. In summary, this study identified genes, whose expression levels may serve as diagnostic markers in MPD.