In silico predicted compound targeting the IQGAP1-GRD domain selectively inhibits growth of human acute myeloid leukemia.

Acute myeloid leukemia (AML) is fatal in the majority of adults. Identification of new therapeutic targets and their pharmacologic modulators are needed to improve outcomes. Previous studies had shown that immunization of rabbits with normal peripheral WBCs that had been incubated with fluorodinitrobenzene elicited high titer antibodies that bound to a spectrum of human leukemias. We report that proteomic analyses of immunoaffinity-purified lysates of primary AML cells showed enrichment of scaffolding protein IQGAP1. Immunohistochemistry and gene-expression analyses confirmed IQGAP1 mRNA overexpression in various cytogenetic subtypes of primary human AML compared to normal hematopoietic cells. shRNA knockdown of IQGAP1 blocked proliferation and clonogenicity of human leukemia cell-lines. To develop small molecules targeting IQGAP1 we performed in-silico screening of 212,966 compounds, selected 4 hits targeting the IQGAP1-GRD domain, and conducted SAR of the 'fittest hit' to identify UR778Br, a prototypical agent targeting IQGAP1. UR778Br inhibited proliferation, induced apoptosis, resulted in G2/M arrest, and inhibited colony formation by leukemia cell-lines and primary-AML while sparing normal marrow cells. UR778Br exhibited favorable ADME/T profiles and drug-likeness to treat AML. In summary, AML shows response to IQGAP1 inhibition, and UR778Br, identified through in-silico studies, selectively targeted AML cells while sparing normal marrow.

Myelodysplastic syndromes disable human CD271+VCAM1+CD146+ niches supporting normal hematopoietic stem/progenitor cells.

Mesenchymal stem/stromal cells (MSCs) within the bone marrow microenvironment (BMME) support normal hematopoietic stem and progenitor cells (HSPCs). However, the heterogeneity of human MSCs has limited the understanding of their contribution to clonal dynamics and evolution to myelodysplastic syndromes (MDS). We combined three MSC cell surface markers, CD271, VCAM-1 (Vascular Cell Adhesion Molecule-1) and CD146, to isolate distinct subsets of human MSCs from bone marrow aspirates of healthy controls (Control BM). Based on transcriptional and functional analysis, CD271+CD106+CD146+ (NGFR+/VCAM1+/MCAM+/Lin-; NVML) cells display stem cell characteristics, are compatible with murine BM-derived Leptin receptor positive MSCs and provide superior support for normal HSPCs. MSC subsets from 17 patients with MDS demonstrated shared transcriptional changes in spite of mutational heterogeneity in the MDS clones, with loss of preferential support of normal HSPCs by MDS-derived NVML cells. Our data provide a new approach to dissect microenvironment-dependent mechanisms regulating clonal dynamics and progression of MDS.

A phase II study of sequential decitabine and rapamycin in acute myelogenous leukemia.

A phase II study was conducted to ascertain whether sequential exposure to decitabine followed by rapamycin, an mTOR (mechanistic target of rapamycin) inhibitor would result in better responses than decitabine alone. Newly diagnosed acute myelogenous leukemia (AML) patients who were >65 years old and not eligible for intensive induction regimens or patients with relapsed or refractory AML received 10 days of decitabine followed by 12 days of rapamycin in cycle 1 and 5 days of decitabine followed by 17 days of rapamycin in subsequent cycles. The composite complete remission rate (CR) was 33 % (CR plus CR with incomplete count recovery). Median overall survival was 7.7 months in newly diagnosed elderly AML patients and 6.6 months in relapsed/refractory AML patients. Twenty-four evaluable patients were enrolled, and the study did not meet its primary endpoint of demonstrating a significant improvement in composite CR rate with the combination as compared to an established historical CR rate of 25 % with decitabine alone. Despite that, the survival rates in relapsed/refractory cases appear comparable to what is reported with other salvage regimens, and no significant patterns of non-hematologic toxicity were noted. 50 % of subjects in the de novo group achieved a composite CR which is significantly higher (p = 0.02) than the rate of 25 % with decitabine alone. This trial is registered at clinical trials.gov as NCT02109744.

Bone marrow mesenchymal stromal cells from acute myelogenous leukemia patients demonstrate adipogenic differentiation propensity with implications for leukemia cell support.

Bone marrow mesenchymal stromal cells (MSCs) constitute one of the important components of the hematopoietic microenvironmental niche. In vivo studies have shown that depletion of marrow MSCs resulted in reduction of hematopoietic stem cell content, and there is in vitro evidence that marrow MSCs are able to support leukemia progenitor cell proliferation and survival and provide resistance to cytotoxic therapies. How MSCs from leukemia marrow differ from normal counterparts and how they are influenced by the presence of leukemia stem and progenitor cells are still incompletely understood. In this work, we compared normal donor (ND) and acute myelogenous leukemia (AML) derived MSCs and found that AML-MSCs had increased adipogenic potential with improved ability to support survival of leukemia progenitor cells. To identify underlying changes, RNA-Seq analysis was performed. Gene ontology and pathway analysis revealed adipogenesis to be among the set of altered biological pathways dysregulated in AML-MSCs as compared with ND-MSCs. Expression of both SOX9 and EGR2 was decreased in AML-MSCs as compared with ND-MSCs. Increasing expression of SOX9 decreased adipogenic potential of AML-MSCs and decreased their ability to support AML progenitor cells. These findings suggest that AML-MSCs possess adipogenic potential which may enhance support of leukemia progenitor cells.

Effects of Neddylation and mTOR Inhibition in Acute Myelogenous Leukemia.

Acute myelogenous leukemia (AML) is a heterogeneous disease and often relapses after standard chemotherapy. Recently, the neddylation (NEDD8) and the mammalian target of rapamycin (mTOR) signaling pathways have emerged as promising pharmaceutical targets for AML therapy. However, the interaction of these two pathways remains unclear. Here we evaluated the effects of pevonedistat, an inhibitor of the NEDD8 activating enzyme (NAE), and sapanisertib (TAK-228), an inhibitor of mTORC1 and mTORC2 as single agents or in combination on AML cell lines. We found that inhibition of neddylation with pevonedistat partially inhibited mTOR signaling transduction and vice versa, inhibition of mTOR signaling with sapanisertib partially inhibited neddylation in AML cell lines. Pevonedistat alone was able to induce cytotoxicity in most AML cell lines as well as in primary AML, whereas sapanisertib alone decreased cell metabolic activity, reduced cell size and arrested cells in G0 phase with only minimal induction of cell death. In addition, pevonedistat was able to induce cell differentiation, arrest cells in G2/M cell cycle phases, and induce DNA re-replication and damage. However, co-treatment with sapanisertib suppressed pevonedistat induced apoptosis, differentiation, S/G2/M arrest, and DNA damage. Taken together, our data demonstrate that pevonedistat and sapanisertib exhibit distinct anti-tumor effects on AML cells, i.e. cytotoxic and cytostatic effects, respectively; however, sapanisertib can attenuate pevonedistat-induced cellular responses in AML cells. Understanding mTOR and neddylation pathway interaction could provide therapeutic strategies for treatment of AML and other malignancies.

Follicular Lymphoma Tregs Have a Distinct Transcription Profile Impacting Their Migration and Retention in the Malignant Lymph Node.

We have previously shown that regulatory T cells (Tregs) infiltrating follicular lymphoma lymph nodes are quantitatively and qualitatively different than those infiltrating normal and reactive nodes. To gain insight into how such Treg populations differ, we performed RNA sequence (RNAseq) analyses on flow sorted Tregs from all three sources. We identify several molecules that could contribute to the observed increased suppressive capacity of follicular lymphoma nodal tregs, including upregulation of CTLA-4, IL-10, and GITR, all confirmed by protein expression. In addition, we identify, and confirm functionally, a novel mechanism by which Tregs target to and accumulate within a human tumor microenvironment, through the down regulation of S1PR1, SELL (L-selectin) and CCR7, potentially resulting in greater lymph node retention. In addition we identify and confirm functionally the upregulation of the chemokine receptor CXCR5 as well as the secretion of the chemokines CXCL13 and IL-16 demonstrating the unique ability of the follicular derived Tregs to localize and accumulate within not only the malignant lymph node, but also localize and accumulate within the malignant B cell follicle itself. Such findings offer significant new insights into how follicular lymphoma nodal Tregs may contribute to the biology of follicular lymphoma and identify several novel therapeutic targets.

Phenotypic, genotypic, and functional characterization of normal and acute myeloid leukemia-derived marrow endothelial cells.

In addition to participation in homing, egress, and transmigration of hematopoietic cells, marrow endothelium also contributes to cell proliferation and survival. Endothelial cells from multiple vascular beds are able to prevent spontaneous or therapy-induced apoptosis in acute myelogenous leukemia (AML) blasts. Marrow-derived endothelial cells from leukemia patients have not been well-characterized, and in this work, endothelial cells were purified from marrow aspirates from normal subjects or from newly diagnosed AML patients to compare these cells phenotypically and functionally. By reverse transcription polymerase chain reaction, these cells express CD31, Tie-2, vascular endothelial growth factor (VEGF), and endothelial nitric oxide synthase (eNOS), supporting endothelial origin. They take up acetyl low-density lipoprotein and are able to form tubular structures. Culture of AML cells with endothelial cells from both normal and AML subjects supported adhesion, transmigration, and leukemia colony-forming unit outgrowth. RNA-sequencing analysis revealed 130 genes significantly up- or downregulated in AML-derived endothelial cells as compared with those derived from normal marrow. The genes differentially expressed (p < 0.001) were included in biological function categories involving cancer, cell development, cell growth and proliferation, cell signaling, inflammatory response, and cell death and survival. Further pathway analysis revealed upregulation of c-Fos and genes involved in chemotaxis such as CXCL16. AML-derived endothelial cells are similar in phenotype and function to their normal marrow-derived counterparts, but genomic analysis suggests a differential signature with altered expression of genes, which could play a role in leukemogenesis or leukemia cell maintenance in the marrow microenvironment.

CD4+ T cell effects on CD8+ T cell location defined using bioluminescence.

T lymphocytes of the CD8+ class are critical in delivering cytotoxic function and in controlling viral and intracellular infections. These cells are "helped" by T lymphocytes of the CD4+ class, which facilitate their activation, clonal expansion, full differentiation and the persistence of memory. In this study we investigated the impact of CD4+ T cells on the location of CD8+ T cells, using antibody-mediated CD4+ T cell depletion and imaging the antigen-driven redistribution of bioluminescent CD8+ T cells in living mice. We documented that CD4+ T cells influence the biodistribution of CD8+ T cells, favoring their localization to abdominal lymph nodes. Flow cytometric analysis revealed that this was associated with an increase in the expression of specific integrins. The presence of CD4+ T cells at the time of initial CD8+ T cell activation also influences their biodistribution in the memory phase. Based on these results, we propose the model that one of the functions of CD4+ T cell "help" is to program the homing potential of CD8+ T cells.

CD8+ T cell immunity to 2009 pandemic and seasonal H1N1 influenza viruses.

A novel strain of H1N1 influenza A virus (pH1N1) emerged in 2009, causing a worldwide pandemic. Several studies suggest that this virus is antigenically more closely related to human influenza viruses that circulated prior to 1957 than viruses of more recent seasonal influenza varieties. The extent to which individuals who are naïve to the 2009 pH1N1 virus carry cross-reactive CD8+ T cells is not known, but a certain degree of reactivity would be expected since there is substantial conservation among the internal proteins of the virus. In the present study, we examined the production of multiple cytokines in response to virus from CD8+ T cells in healthy adult subjects, between 18 and 50 years of age (born post 1957), who had no evidence of exposure to the 2009 pH1N1 virus, and had blood collected prior to the emergence of the pandemic in April of 2009. Human peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with a panel of live viruses, and assayed by intracellular cytokine staining and flow cytometry. Although results were variable, most subjects exhibited cytokine positive CD8+ T cells in response to pH1N1. Cytokine producing cells were predominantly single positive (IL2, IFNγ, or TNFα); triple-cytokine producing cells were relatively rare. This result suggests that although many adults carry cross-reactive T cells against the emergent pandemic virus, these cells are in a functionally limited state, possibly because these subjects have not had recent exposure to either seasonal or pandemic influenza strains.

Galactosylated LDL nanoparticles: a novel targeting delivery system to deliver antigen to macrophages and enhance antigen specific T cell responses.

We aim to define the role of Kupffer cells in intrahepatic antigen presentation, using the selective delivery of antigen to Kupffer cells rather than other populations of liver antigen-presenting cells. To achieve this we developed a novel antigen delivery system that can target antigens to macrophages, based on a galactosylated low-density lipoprotein nanoscale platform. Antigen was delivered via the galactose particle receptor (GPr), internalized, degraded and presented to T cells. The conjugation of fluoresceinated ovalbumin (FLUO-OVA) and lactobionic acid with LDL resulted in a substantially increased uptake of FLUO-OVA by murine macrophage-like ANA1 cells in preference to NIH3T3 cells, and by primary peritoneal macrophages in preference to primary hepatic stellate cells. Such preferential uptake led to enhanced proliferation of OVA specific T cells, showing that the galactosylated LDL nanoscale platform is a successful antigen carrier, targeting antigen to macrophages but not to all categories of antigen presenting cells. This system will allow targeted delivery of antigen to macrophages in the liver and elsewhere, addressing the question of the role of Kupffer cells in liver immunology. It may also be an effective way of delivering drugs or vaccines directly at macrophages.

Imaging CD8+ T cell dynamics in vivo using a transgenic luciferase reporter.

After activation, populations of antigen-specific T cells flow between sites of antigen expression, local lymphoid structures and other lymphoid and non-lymphoid organs. In this study, we documented the in vivo dynamics of a CD8(+) T cell response to antigen delivered using herpes simplex virus amplicon vectors and revealed several unexpected features. First, the T cells localized to the site of vector injection, as well as the draining lymph node within 24-48 h. Second, the major site to which T cells later redistributed were intra-abdominal lymphoid organs, including milky spots, mesenteric and lumbar lymph nodes. We determined the relationship between bioluminescent signal and antigen-specific T cell numbers in various lymphoid organs, and concluded that bioluminescent signal is a valid surrogate measure of T cell abundance in superficial lymph nodes, but not in deeper structures such as the spleen.

The E2F-1 transcription factor promotes caspase-8 and bid expression, and enhances Fas signaling in T cells.

The immune system depends on the extensive proliferation of rare Ag-specific precursor T lymphocytes, followed by their differentiation, the delivery of effector function, and finally death by apoptosis. T cells that lack the E2F-1 transcription factor, which is activated as cells pass the restriction point and enter S phase, show defects in activation-induced cell death. We now report that E2F-1 increases the activity of an apoptotic pathway that is important in murine primary T cells. Thus, E2F-1 promotes the transcription of Bid, a molecule that links death receptor signaling to the activation of apoptotic mechanisms in mitochondria. It also promotes the transcription of caspase-8, the enzyme that cleaves and activates Bid. Enforced expression of Bid can partially restore apoptosis in E2F-1-deficient T cells. Thus, E2F-1 integrates cell cycle progression with apoptosis.

Systemic effects of inhaled ultrafine particles in two compromised, aged rat strains.

Epidemiological studies associate morbidity and mortality with exposure to particulate air pollution in elderly individuals with existing cardiopulmonary disease. These associations led to the hypothesis that inhaled particles can exert adverse effects outside of the lung, particularly on the cardiovascular system. We tested this hypothesis by examining the pulmonary and peripheral effects of inhaled ultrafine carbon particles in old rats that were injected with endotoxin (lipopolysaccharide, LPS) to model systemic gram-negative bacterial infection. Fischer 344 rats (23 mo) and spontaneously hypertensive (SH) rats (11-14 mo) were injected with LPS (2 mg/kg, i.p.) immediately before being exposed to inhaled ultrafine carbon particles for 6 h (150 microg/m(3), CMD = 36 nm). Controls were injected with sterile saline or were sham exposed. Twenty-four hours after LPS injection, bronchoalveolar lavage (BAL) fluid, cells, and blood were obtained to assess endpoints of inflammation, oxidant stress, coagulability, and the acute-phase response. LPS did not cause an influx of neutrophils (PMNs) into the alveolar space, but did increase the number and percentage of circulating PMNs and the concentration of plasma fibrinogen in both rat strains. Inhaled ultrafine particles did not induce lung inflammation in either rat strain. In both strains, ultrafine particles (UFP) were found to decrease the number of blood PMNs, increase the intracellular oxidation of a fluorescent dye (DCFD) in blood PMNs, and affect plasma thrombin-anti-thrombin (TAT) complex and fibrinogen levels. UFP were also found to interact with ip LPS with respect to plasma TAT complex levels and blood PMN DCFD oxidation. Differences between the two rat strains were also found for TAT complex levels, BAL cell reactive oxygen species release, and DCFD oxidation in both BAL macrophages and blood PMNs. These results suggest that inhaled ultrafine carbon particles inhaled at concentrations mimicking high episodic increases in urban air can exert extrapulmonary effects in old rats and that they can change the systemic response to an inflammatory stimulus.

Nitrogen dioxide exposure: effects on airway and blood cells.

This study examined the effects of nitrogen dioxide (NO(2)) exposure on airway inflammation, blood cells, and antiviral respiratory defense. Twenty-one healthy volunteers were exposed on separate occasions to air and 0.6 and 1.5 ppm NO(2) for 3 h with intermittent moderate exercise. Phlebotomy and bronchoscopy were performed 3.5 h after each exposure, and recovered cells were challenged with respiratory viruses in vitro. Blood studies revealed a 4.1% NO(2) dose-related decrease in hematocrit (P = 0.003). Circulating total lymphocytes (P = 0.024) and T lymphocytes (P = 0.049) decreased with NO(2) exposure. Exposure to NO(2) increased the blood lymphocyte CD4(+)-to-CD8(+) ratio from 1.74 +/- 0.11 to 1.85 +/- 0.12 in males but decreased it from 1.88 +/- 0.19 to 1.78 +/- 0.19 in females (P < 0.001 for gender difference). Polymorphonuclear leukocytes in bronchial lavage increased with NO(2) exposure (P = 0.003). Bronchial epithelial cells obtained after exposure to 1.5 ppm NO(2) released 40% more lactate dehydrogenase after challenge with respiratory syncytial virus than with air exposure (P = 0.024). In healthy subjects, exposures to NO(2) at levels found indoors cause mild airway inflammation, effects on blood cells, and increased susceptibility of airway epithelial cells to injury from respiratory viruses.

Temporality in Ultrasound-Induced Cell Lysis In Vitro.

To test the hypothesis that the full extent of in vitro cell lysis due to ultrasound becomes evident with time lapse following insonation, human erythrocytes (2% hematocrit) in autologous plasma were mixed with Albunex(R), a pulse echo contrast agent, and exposed to 1-MHz, continuous-wave ultrasound (US) (5 W/cm(2) SPTA intensity) for 60 seconds while in a rotating (200 rpm) dialysis membrane vessel. Exposed and sham-exposed samples were subsequently assayed for hemolysis colorimetrically, either immediately or after a delay of 3 hours. Hemolysis was dependent on the interval between US exposure and assay, with significantly greater lysis evident with delayed assay. There was also temporality in lytic yield with sample number, i.e., with time postpreparation of the blood sample, US-induced cell lysis decreased. The temporality of lytic yield was eliminated by maintenance at ice water temperatures, or by waiting about 1 hour before beginning treatments. The collective data indicate that the full extent of US-induced cell lysis is not evident upon assay immediately after insonation, and that with time postpreparation and preinsonation, erythrocytes may undergo changes in sensitivity to US. (ECHOCARDIOGRAPHY, Volume 13, January 1996)