ALBERT

Description: Insulin-like growth factor binding protein-3 (IGFBP-3) can cause growth suppressive and proapoptotic effects on retinoids in many types of cancer cells. However, the expression and effects of IGFBP-3 in myeloid leukemia cells have not been elucidated. In this study, we found no IGFBP-3 expression in the human myeloid leukemia cell lines either at baseline or after stimulation with all-trans retinoic acid (ATRA). Human recombinant IGFBP-3 induced growth arrest and apoptosis of HL-60 and NB4 cells. We have previously identified RXRα as a nuclear receptor for IGFBP-3 and have proceeded to examine further the role of this interaction in leukemia cell lines. In signaling assays, IGFBP-3 potently suppressed RAR- and VDR-mediated signaling while enhancing RXR signaling. Interestingly, when IGFBP-3 was administered to these cells in combination with an RAR-selective ligand, the ability of these retinoids to induce differentiation was blunted. On the other hand, IGFBP-3 enhanced the effect of an RXR-selective ligand to induce differentiation of HL-60 and NB4 cells. Further studies showed that IGFBP-3 down-regulated (at the transcriptional level) the retinoid-induced expression of C/EBPϵ in NB4 cells. Taken together, these results indicate that IGFBP-3 has antiproliferative activity against myeloid leukemia cells; while it enhances signaling through RXR/RXR, it blunts signaling by activated RAR/RXR.

Description: C/EBPδ belongs to the family of highly conserved CCAAT/enhancer binding protein (C/EBP) transcription factors. Members of this family play a critical role in the regulation of mitotic growth arrest and differentiation in numerous cell types. To examine the consequences of C/EPBδ expression, we transfected C/EPBδ into CML myeloid leukemia (KCL22, K562), prostate (LNCaP, PC3, DU145), and breast (MCF-7, T47D, MDA-MB-231) cancer cell lines. C/EBPδ expression resulted in a proliferative arrest and an increase in apoptosis of the myeloid leukemia cells, as well as the prostate cells LNCaP and PC3, and the breast cells MCF-7 and T47D. In contrast, DU145 prostate and MDA-MB-231 breast cancer cells were not inhibited by C/EBPδ, indicating that the biologically properties of C/EBPδ depend upon its cellular context. We further studied the molecular mechanisms underlying the affect of C/EPBδ expression in CML leukemic cells. Myeloid differentiation of KCL22 and K562 blast cells as shown by morphologic changes and induction of secondary specific granule genes, occurred within 4 days of inducing expression of C/EBPδ. Furthermore, expression of C/EBPδ was associated with downregulation of c-Myc and cyclin E, and upregulation of the forkhead transcription factor FoxO1a (FKHR) and the cyclin-dependent kinase inhibitor p27Kip1. In addition, microarray analysis showed that C/EBPδ mRNA is upregulated during granulocytic differentiation of normal CD34+ bone marrow cells, suggesting that C/EBPδ is involved in lineage-specific differentiation. Taken together, these results show that expression of C/EBPδ in BCR-ABL-positive CML cells in blast crisis, is sufficient for neutrophil differentiation and suggest that ectopic induction of C/EBPδ in the blastic phase of CML, as well as in certain cases of prostate and breast cancers, may hold promising therapeutic potential.

Description: Ritonavir (RTV) is a potent inhibitor of cytochrome p450 (CYPs) enzymes. This study explores the effects of RTV on CYP24 which converts 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] to its inactive form 1,24,25,(OH)3. Real-time RT-PCR showed that exposure of HL-60 cells to 1,25(OH)2D3 (10−7 M, 1h) induced expression of CYP24 by 80-fold, and pre-incubation of these cells with RTV (2X10−5 M, 3h) decreased 1,25(OH)2D3-stumulated expression of CYP24 transcripts by 30 %, resulting in increased levels of 1,25(OH)2D3 in culture media (50.7 vs 82.4 nmol/L). Importantly, pre-incubation of HL-60 cells with RTV (2X10−5 M, 3h) and then exposure to 1,25(OH)2D3 (10−7 M, 24 h) also increased intracellular levels of 1,25(OH)2D3 (1.20 vs 2.46 nmol/L) and potentiated the ability of 1,25(OH)2D3 to induce growth arrest of HL-60 cells. Clonogenic assay showed that either RTV (5X10−6 M) or 1,25(OH)2D3 (10−8 M) alone inhibited colonal growth of HL-60 cells by either a mean 30 ± 12 (±SD) % and 40 ± 12 %, respectively; when both were combined, colony formation was inhibited by a mean 80 ± 13 % (p

Description: This study found that MS-275, a novel synthetic benzamide histone deacetylase inhibitor (HDACI), blocked Akt/mTOR signaling in acute myelogenous leukemia (AML) HL60 and acute promyelocytic leukemia (APL) NB4 cells, as assessed by decreased levels of the phosphorylated (p)-Akt, p-p70 ribosomal S6 kinase (p70S6K), and p-S6K by Western blot analysis. Interestingly, further inactivation of mTOR by rapamycin analogue RAD001 (everolimus) significantly enhanced MS-275-mediated growth inhibition and apoptosis of these cells in parallel with enhanced upregulation of p27 kip1 and downregulation of c-Myc. In addition, RAD001 potentiated the ability of MS-275 to induce differentiation of HL60 and NB4 cells, as measured by expression of CD11b cell surface antigens, as well as reduction of nitroblue tetrazolium. Importantly, RAD001 potentiated the ability of MS-275 to induce expression of the myeloid differentiation-related transcription factor CCAAT enhancer binding protein e in these cells in association with enhanced acetylation of histone H3 on its promoter. Furthermore, RAD001 (5 mg/kg) significantly enhanced the effects of MS-275 (10 mg/kg) to inhibit proliferation of HL60 tumor xenografts in nude mice without adverse effects. Taken together, concomitant administration of a HDACI and a mTOR inhibitor may be a promising treatment strategy for the individuals with a subset of human leukemia.

Description: Abstract 2168 To identify molecular targets in leukemia stem cells (LSCs), this study compared the protein expression profile of freshly isolated LSCs (CD34+/CD38- compartment) with that of non-LSC (CD34+/CD38+ compartment) counterparts from individuals with acute myelogenous leukemia (AML) using isobaric tags for relative and absolute quantitation (iTRAQ). A total of 98 proteins were overexpressed, while six proteins were underexpressed in LSCs compared with their non-LSC counterparts. Proteins overexpressed in LSCs included a number of proteins involved in DNA repair, cell cycle arrest, gland differentiation, anti-apoptosis, adhesion, and drug resistance. Aberrant expression of CD82, a family of adhesion molecules, in LSCs was noted in additional clinical samples (n=6) by flow cytometry. In addition, we found that imatinib-resistant chronic eosinophilic leukemi EOL-1R cells expressed a greater amount of CD82 and remained in a dormant state compared to the parental EOL-1 cells. Interestingly, down-regulation of CD82 in EOL-1R cells by a small interfering RNA stimulated their migration capacity, as assessed by the transwell assay. These observations suggested that the aberrant expression of CD82 probably played a role in adhesion of hematopoietic cells to bone marrow microenvironment. Targeting CD82 could detach LSCs from bone marrow niche and sensitized these cells to anti-leukemia agents. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1886 Aurora kinase A (AURKA) plays a pivotal role in the mitotic processes during cell division. We previously showed that AURKA was aberrantly expressed in hematological malignant cells including those from acute myelogenous leukemia (AML) and acute lymphoblastic leukemia, compared with CD34+ hematopoietic stem/progenitor cells isolated from healthy volunteers. This study found that freshly isolated CD34+/CD38− cells from individuals with AML (n=12) in which leukemia stem cells were supposed to be enriched expressed a greater amount of AURKA than their CD34+/CD38+ counterparts, as measured by real time RT-PCR. Blockade of AURKA by the specific inhibitor MLN8237 significantly inhibited proliferation and induced apoptosis of CD34+/CD38− AML cells, as assessed by the clonogenic assay and detection of the cleaved form of poly (ADP-ribose) polymerase by Western blot analysis, respectively. In addition, exposure of CD34+/CD38− AML cells to MLN8237 down-regulated levels of Bcl-2 family proteins and increased Bax/Bcl-2 expression ratio. Moreover, inhibition of AURKA by MLN8237 (30mg/kg for 14 consecutive days) impaired engraftment of CD34+/CD38− AML cells in severely immunocompromised mice (n=5, 49±32% in control vs 18±16% in MLN8237 treated mice, P

Description: Inhibitors of the protease of human immunodeficiency virus type 1 (HIV-1) may inhibit cytoplasmic retinoic acid-binding proteins, cytochrome P450 isoforms, as well as P-glycoproteins. These features of the protease inhibitors might enhance the activity of retinoids. To explore this hypothesis, myeloid leukemia cells were cultured with all-trans retinoic acid (ATRA) either alone or in combination with the HIV-1 protease inhibitors indinavir, ritonavir, and saquinavir. Consistent with the hypothesis, the HIV-1 protease inhibitors enhanced the ability of ATRA to inhibit growth and induce differentiation of HL-60 and NB4 myeloid leukemia cells, as measured by expression of CD11b and CD66b cell surface antigens, as well as reduction of nitroblue tetrazolium. Growth of ATRA-resistant UF-1 cells was also inhibited when cultured with the combination of ATRA and indinavir. Moreover, indinavir enhanced the ability of ATRA to induce expression of the myeloid differentiation-related transcription factor C/EBPε messenger RNA in NB4 cells by 9.5-fold. Taken together, the results show that HIV-1 protease inhibitors enhance the antiproliferative and differentiating effects of ATRA on myeloid leukemia cells. An HIV-1 protease inhibitor might be a useful adjuvant with ATRA for patients with acute promyelocytic leukemia and possibly retinoid-resistant cancers.

Description: Abstract 2981 We recently reported that adhesion molecule CD82 is aberrantly expressed in CD34+/CD38− leukemia stem cells (LSCs) (ASH 2010; Abstract 2168.). Here, we report the results of a functional analysis of CD82 in CD34+/CD38− acute myelogenous leukemia (AML) cells. Short hairpin (sh)RNA-mediated downregulation of CD82 resulted in dephosphorylation of STAT5 and a decrease in the level of IL-10. In contrast, forced expression of CD82 in CD34+/CD38+ AML cells by transduction with CD82-expressing lentiviral particles resulted in an increase in the levels of p-STAT5 and IL-10. Chromatin immunoprecipitation (ChIP) assay results indicated that STAT5A binds to the promoter region of the IL-10 gene, while reporter gene assay results indicated stimulation of IL-10 expression at the transcriptional level. These results suggest that CD82 positively regulates the STAT5/IL-10 signaling pathway. Notably, exposure of CD34+/CD38− AML cells to IL-10 stimulated clonogenic growth of these cells. Moreover, shRNA-mediated downregulation of CD82 expression in CD34+/CD38− AML cells impaired AML reconstitution in immunodeficient mice in parallel with downregulation of STAT5. Taken together, our data suggest that the CD82/STAT5/IL-10 signaling pathway is involved in the survival of CD34+/CD38− AML cells and may thus be a promising therapeutic target for eradication of AML LSCs. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Thrombotic microangiopathy (TMA) and sinusoidal obstruction syndrome (SOS) as well as acute GVHD are major complications after allogeneic hematopoietic stem cell transplantation (alloHSCT) to overcome to achieve better overall survival. These complications are closely relevant to inflammation and coagulation involving endothelium, which are named as transplantation-associated coagulopathy (TAC). Recombinant thrombomodulin (rTM) is a new drug for treating DIC approved by Japanese Ministry of Health, Labour and Welfare in 2008. Membranous TM has many aspects to inhibit inflammation via activated Protein C (APC), lectin-like domain, and activated thrombin-activatable fibrinolysis inhibitor as well as to inhibit coagulation by binding factor IXa and Xa through APC. Biomarkers such as inflammatory cytokines and endothelial molecules are expected to be clue in elucidating TAC. We established SIGHT study group to explore above complications following alloHSCT. In the present study, we investigated the effects of rTM on levels of the biomarkers and whether we could prevent TAC using rTM among registered patients in the SIGHT study group. Patients and methods: SIGHT study group covered nationwide institutes more than 70 in Japan. Patients whoever were eligible for alloHSCT to treat hematopoietic disorders by physicians decision were permitted and written informed consent was requested to register in the study. Blood samples were collected from the patients before and after transplantation through day 28. Levels of cytokines (IL-6, TNF-α, HMGB1, MCP-1, RANTES) and soluble molecules (VCAM-1, E-selectin, PAI-1, PDMP) were measured by ELISA. rTM was administered as a prophylactic therapy for TAC in day 4-14 after alloHSCT. Control group was received heparin or no anti-coagulation therapy as prophylaxis during same schedule as rTM group. Patients were not randomized to these three arms but allowed to select one by their physicians decision. The primary endpoint was a comparison of fluctuation of these biomarkers in three arms. Secondary endopoint was a comparison of the rate of developing TAC. Results: The subjects were 293 patients who underwent alloHSCT in SIGHT study group between June 2010 and February 2014. 271 patients out of them were analyzed to measure level of these biomarkers. There was no significant difference in all characteristics but GVHD prophylaxis between two groups. rTM group received more cyclosporine than control group (p=0.05). MCP-1 and IL-6 exhibited more significant elevations on day 7 after alloHSCT, while HMGB-1 did on the day of alloHSCT. In contrast, the levels of TNF-α, E-selectin, VCAM-1, PAI-1 and PDMP exhibited increment since around day 7 after alloHSCT. Significant improvements in TNF-α, E-selectin, VCAM-1, HMGB-1, PAI-1 and PDMP was shown after rTM-treatment, but not shown in control group. Regarding complications following alloHSCT, the rate of developing acute GVHD and SOS was significantly lower in rTM group than without it (36.5% vs 62.2%, p=0.000; 12.5% vs 24.5%, p=0.032, respectively), while TMA did not differ (8.4% vs 10.2%, p=0.961). Stepwise multivariate logistic regression analyses revealed that anti-coagulation therapy without rTM and the level of PAI-1 on day 28 after HSCT were independent risk factor for acute GVHD (p=0.000, odds ratio=3.006; p=0.000, odds ratio=1.068, respectively). Also anti-coagulation therapy without rTM and the level of HMGB-1 on day 28 after HSCT were significantly predictive for risk of SOS (p=0.015, odds ratio=2.650; p=0.001, odds ratio=1.433, respectively). Tacrolimus was significantly good risk of SOS (p=0.022, odds ratio=0.404). The level of VCAM-1 on day 28 after HSCT was significantly associated with risk of TMA (p=0.040, odds ratio=1.001). Conclusions: We identified predictive biomarkers for TAC following alloHSCT, which were PAI-1 for acute GVHD, HMGB-1 for SOS, and VCAM-1 for TMA, respectively in this study. The present findings suggest that rTM has the possibility to play a therapeutic role for acute GVHD and SOS induced by suppression on these biomarkers. Endothelial and pro-coagulant biomarkers were released delayed compared with inflammatory biomarkers. TMA is reported to develop around day 44 in median after alloHSCT. Further studies are needed whether rTM should administer later or longer to improve TMA than the present duration after alloHSCT. Disclosures No relevant conflicts of interest to declare.