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BACKGROUND: Resistance to imatinib monotherapy frequently emerges in advanced stages of chronic myelogenous leukemia (CML), supporting the rationale for combination drug therapy. In the present study, the activities of the farnesyltransferase inhibitors (FTIs) L744,832 and LB42918, as single agents and in combination with imatinib, were investigated in different imatinib-sensitive and -resistant BCR-ABL-positive CML cells. MATERIALS AND METHODS: Growth inhibition of the cell lines and primary patient cells was assessed by MTT assays and colony-forming cell assays, respectively. Drug interactions were analyzed according to the median-effect method of Chou and Talalay. The determination of apoptotic cell death was performed by annexin V/propidium iodide staining. RESULTS: Combinations of both FTIs with imatinib displayed synergism or sensitization (potentiation) in all the cell lines tested. In primary chronic phase CML cells, additive and synergistic effects were discernible for the combination of imatinib plus L744,832 and imatinib plus LB42918, respectively. Annexin V/propidium iodide staining showed enhancement of imatinib-induced apoptosis with either drug combination, both in imatinib-sensitive and -resistant cells. CONCLUSION: The results indicated the potential of L744,832 and LB42918 as combination agents for CML patients on imatinib treatment.

Adenosine deaminase (ADA) deficiency is caused by a purine metabolic dysfunction, leading to severe combined immunodeficiency (SCID) and multiple organ damage. To investigate the efficacy of ex vivo gene therapy with self-inactivating lentiviral vectors (LVs) in correcting this complex phenotype, we used an ADA(-/-) mouse model characterized by early postnatal lethality. LV-mediated ADA gene transfer into bone marrow cells combined with low-dose irradiation rescued mice from lethality and restored their growth, as did transplantation of wild-type bone marrow. Mixed chimerism with multilineage engraftment of transduced cells was detected in the long term in animals that underwent transplantation. ADA activity was normalized in lymphocytes and partially corrected in red blood cells (RBCs), resulting in full metabolic detoxification and prevention of severe pulmonary insufficiency. Moreover, gene therapy restored normal lymphoid differentiation and immune functions, including antigen-specific antibody production. Similar degrees of detoxification and immune reconstitution were obtained in mice treated early after birth or after 1 month of enzyme-replacement therapy, mimicking 2 potential applications for ADA-SCID. Overall, this study demonstrates the efficacy of LV gene transfer in correcting both the immunological and metabolic phenotypes of ADA-SCID and supports the future clinical use of this approach.

The B-cell receptor (BCR) transmits life and death signals throughout B-cell development, and altered BCR signaling may be required for survival of B-lymphoma cells. We used single-cell signaling profiles to compare follicular lymphoma (FL) B cells and nonmalignant host B cells within individual patient biopsies and identified BCR-mediated signaling events specific to lymphoma B cells. Expression of CD20, Bcl-2, and BCR light chain isotype (kappa or lambda) distinguished FL tumor B-cell and nontumor host B-cell subsets within FL patient biopsies. BCR-mediated signaling via phosphorylation of Btk, Syk, Erk1/2, and p38 occurred more rapidly in tumor B cells from FL samples than in infiltrating nontumor B cells, achieved greater levels of per-cell signaling, and sustained this level of signaling for hours longer than nontumor B cells. The timing and magnitude of BCR-mediated signaling in nontumor B cells within an FL sample instead resembled that observed in mature B cells from the peripheral blood of healthy subjects. BCR signaling pathways that are potentiated specifically in lymphoma cells should provide new targets for therapeutic attention.

While statin treatment may transiently mobilize endothelial progenitor cells (EPCs), the dose-dependent effects of a continuous statin therapy on EPCs in patients with chronic coronary artery disease (CAD) have not been analyzed. In 209 patients with angiographically documented CAD, 144 of which received 10-40 mg/day of statins for textgreater8 weeks, the EPC number was determined by flow cytometry directly (CD34(+)/KDR(+), n=58) and after in vitro-culture (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled Ac-LDL (DiI-Ac-LDL(+))/lectin(+), n=209). EPC function was assessed by the formation of colony forming units (CFUs). Univariate analysis revealed that the dose of continuous statin therapy inversely correlated with the EPC number. Treatment with 40 mg/day significantly reduced EPC counts. Multivariate analysis unveiled the statin dose and extent of CAD as independent predictors of reduced EPC numbers. Conversely, obesity predicted increased counts, while CFU development was not detectable in all patients and augmented in females and smokers but not in statin-treated patients. Compared with matched controls, statin-treated patients showed significantly reduced absolute and relative EPC counts. In a prospective analysis, initiation of statin therapy significantly diminished the number of circulating and isolated EPCs after 3 but not after 1 month(s). Thus, the statin dose during chronic and continuous treatment independently predicts reduced numbers of circulating as well as isolated EPCs in patients with CAD.

Activating mutations of c-KIT lead to ligand-independent growth. Internal tandem duplications (ITDs) of exon 11, which encodes the juxtamembrane domain (JMD), are constitutively activating mutations found in 7% of gastrointestinal stromal tumors (GISTs) but have not been described in childhood acute myeloid leukemia (AML). DNA and cDNA from 60 children with AML were screened by polymerase chain reaction (PCR) for mutations of the JMD. A complex ITD (kit cITD) involving exon 11 and exon 12 was identified with a relative frequency of 7% (4/60). The human kit cITDs were inserted into the murine c-Kit backbone and expressed in Ba/F3 cells. KIT cITD induced factorindependent growth and apoptosis resistance, and exhibited constitutive autophosphorylation. KIT cITD constitutively activated the PI3K/AKT pathway and phosphorylated STAT1, STAT3, STAT5, and SHP-2. Imatinib (IM) or rapamycin (Rap) led to complete inhibition of growth, with IC50 values at nanomolar levels. IM and Rap synergistically inhibited growth and surmounted KIT cITD-induced apoptosis resistance. IM but not LY294002 inhibited phosphorylation of STAT3 and STAT5, suggesting aberrant cross talk between PI3K- and STAT-activating pathways. The findings presented may have immediate therapeutic impact for a subgroup of childhood AML-expressing c-KIT mutations.

Histone acetyltransferases (HATs) regulate transcription, chromatin structure and DNA repair. Here, we utilized a novel HAT inhibitor, anacardic acid, to examine the role of HATs in the DNA damage response. Anacardic acid inhibits the Tip60 HAT in vitro, and blocks the Tip60-dependent activation of the ATM and DNA-PKcs protein kinases by DNA damage in vivo. Further, anacardic acid sensitizes human tumor cells to the cytotoxic effects of ionizing radiation. These results demonstrate a central role for HATs such as Tip60 in regulating the DNA damage response. HAT inhibitors provide a novel therapeutic approach for increasing the sensitivity of tumors to radiation therapy.

MafB is a member of the large Maf family of transcription factors that share similar basic region/leucine zipper DNA binding motifs and N-terminal activation domains. Although it is well known that MafB is specifically expressed in glomerular epithelial cells (podocytes) and macrophages, characterization of the null mutant phenotype in these tissues has not been previously reported. To investigate suspected MafB functions in the kidney and in macrophages, we generated mafB/green fluorescent protein (GFP) knock-in null mutant mice. MafB homozygous mutants displayed renal dysgenesis with abnormal podocyte differentiation as well as tubular apoptosis. Interestingly, these kidney phenotypes were associated with diminished expression of several kidney disease-related genes. In hematopoietic cells, GFP fluorescence was observed in both Mac-1- and F4/80-expressing macrophages in the fetal liver. Interestingly, F4/80 expression in macrophages was suppressed in the homozygous mutant, although development of the Mac-1-positive macrophage population was unaffected. In primary cultures of fetal liver hematopoietic cells, MafB deficiency was found to dramatically suppress F4/80 expression in nonadherent macrophages, whereas the Mac-1-positive macrophage population developed normally. These results demonstrate that MafB is essential for podocyte differentiation, renal tubule survival, and F4/80 maturation in a distinct subpopulation of nonadherent mature macrophages.

Differences in BCR signaling may govern outcomes as diverse as proliferation and cell death. We profiled BCR signaling kinetics in subsets of primary human B cells using flow cytometry. In the predominant population expressing IgM, BCR cross-linking led to a quick burst of Syk, ERK1/2, and p38 signaling. In contrast, IgG B cells sustained higher per-cell ERK1/2 phosphorylation over time. This dichotomy suggested a mechanism for dampening signals transmitted by IgM. Regulatory phosphatase activity in IgM B cells was BCR-mediated and initiated more slowly than kinase activity. This BCR-mediated phosphatase activity was sensitive to inhibition by H(2)O(2) and required to attenuate IgM BCR signaling. These results provide the first kinetic maps of BCR signaling in primary human B cell subsets and enable new studies of signaling in B cell disorders, such as autoimmunity and cancer.

The purpose of these studies is to determine why an immunogenic tumor grows unchecked in the anterior chamber (a.c.) of the eye. The OVA-expressing EL4 tumor, E.G7-OVA, was injected into the a.c. or skin of immunocompetent and immunodeficient mice. Tumor growth and tumor-specific immune responses were monitored. Ocular tumor-infiltrating leukocytes were characterized phenotypically and functionally. Growth of E.G7-OVA was inhibited when limiting numbers of cells were injected in the skin but not in the a.c. of C57BL/6 mice, although both routes primed OVA-specific immune responses, which prevented the growth of a subsequent injection with E.G7-OVA in the skin or opposite eye. Tumor regression was OVA-specific because growth of the parental EL-4 tumor was not inhibited in primed mice. E.G7-OVA growth in the skin was not inhibited in immunodeficient Rag(-/-) or CD8 T cell-deficient mice, suggesting that CD8(+) CTLs mediate tumor elimination. CD8(+) T cell numbers were significantly increased in eyes of mice primed with E.G7-OVA, but few were detected in primary ocular tumors. Nevertheless, growth of E.G7-OVA was retarded in the a.c. of TCR-transgenic OT-I mice, and CD8(+) T cell numbers were increased within eyes, suggesting that tumor-specific CD8(+) CTLs migrated into and controlled primary ocular tumor growth. E.G7-OVA did not lose antigenicity or become immunosuppressive after 13 days of growth in the eye. However, CD11b(+) cells accumulated in primary ocular tumors and contained potent immunosuppressive activity when assayed in vitro. Thus, CD11b(+) cells that accumulate within the eye as tumors develop in the a.c. may contribute to immune evasion by primary ocular tumors by inhibiting CTLs within the eye.

Stem cell leukemia/T cell acute leukemia 1 (SCL/TAL1) plays a key role in the development of murine primitive hematopoiesis but its functions in adult definitive hematopoiesis are still unclear. Using lentiviral delivery of TAL1-directed shRNA in human hematopoietic cells, we show that decreased expression of TAL1 induced major disorders at different levels of adult hematopoietic cell development. Erythroid and myeloid cell production in cultures was dramatically decreased in TAL1-directed shRNA-expressing cells, whereas lymphoid B-cell development was normal. These results confirm the role of TAL1 in the erythroid compartment and show TLA1's implication in the function of myeloid committed progenitors. Moreover, long-term cultures and transplantation of TAL1-directed shRNA-expressing CD34+ cells into irradiated nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice led to dramatically low levels of human cells of all lineages including the B-lymphoid lineage, strongly suggesting that TAL1 has a role in the early commitment of hematopoietic stem cells (HSCs) in humans. Cultures and transplantation experiments performed with mouse Sca1+ cells gave identical results. Altogether, these observations definitively show that TAL1 participates in the regulation of hematopoiesis from HSCs to myeloid progenitors, and pinpoint TAL1 as a master protein of human and murine adult hematopoiesis.

Aldehyde dehydrogenase (ALDH) is an enzyme that is expressed in the liver and is required for the conversion of retinol (vitamin A) to retinoic acids. ALDH is also highly enriched in hematopoietic stem cells (HSCs) and is considered a selectable marker of human HSCs, although its contribution to stem cell fate remains unknown. In this study, we demonstrate that ALDH is a key regulator of HSC differentiation. Inhibition of ALDH with diethylaminobenzaldehyde (DEAB) delayed the differentiation of human HSCs that otherwise occurred in response to cytokines. Moreover, short-term culture with DEAB caused a 3.4-fold expansion in the most primitive assayable human cells, the nonobese diabetic/severe combined immunodeficiency mouse repopulating cells, compared with day 0 CD34(+)CD38(-)lin(-) cells. The effects of DEAB on HSC differentiation could be reversed by the coadministration of the retinoic acid receptor agonist, all-trans-retinoic acid, suggesting that the ability of ALDH to generate retinoic acids is important in determining HSC fate. DEAB treatment also caused a decrease in retinoic acid receptor-mediated signaling within human HSCs, suggesting directly that inhibition of ALDH promotes HSC self-renewal via reduction of retinoic acid activity. Modulation of ALDH activity and retinoid signaling is a previously unrecognized and effective strategy to amplify human HSCs.

Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy, and clinical transplantation. Here, we demonstrate efficient ex vivo expansion of HSCs measured by long-term severe combined immunodeficient (SCID) repopulating cells (SRCs) from human cord blood CD133-sorted cells using a soluble form of Delta1. After a 3-week culture on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, Flt-3 ligand, interleukin (IL)-3, and IL-6/soluble IL-6 receptor chimeric protein (FP6) in a serum- and stromal cell-free condition, we achieved approximately sixfold expansion of SRCs when evaluated by limiting dilution/transplantation assays. The maintenance of full multipotency and self-renewal capacity during culture was confirmed by transplantation to nonobese diabetic/SCID/gammac(null) mice, which showed myeloid, B, T, and natural killer cells as well as CD133(+)CD34(+) cells, and hematopoietic reconstitution in the secondary recipients. Interestingly, the CD133-sorted cells contained approximately 4.5 times more SRCs than the CD34-sorted cells. The present study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.