IL-3 Dependent Cell Line Research Articles

NOVEL Pathways Modulate TUMOR CELL Susceptibility to NK CELLS.

Abstract 277NK cells play an important role in innate immune responses directed against autologous cells that have undergone viral or malignant transformation. The ability of NK cells to lyse targets is primarily dependent on the expression of various inhibitory or activating receptors. However, transformed cells have also developed mechanisms to evade immune surveillance and the molecular basis for target resistance to immune-mediated lysis is not well understood. To address this issue we undertook a genetic screening approach to identify novel pathways that modulate tumor cell susceptibility to NK cell lysis. Our genetic screen utilized a subset of the TRC1 lentiviral shRNA library developed at the Broad Institute of Harvard and MIT. The library subset targeted 476 protein kinases and 180 phosphatases that represent 88% and 80%, respectively, of known NCBI sequences with these functions. The library also targeted 372 genes representing tumor suppressors, DNA binding proteins as well as irrelevant shRNAs as controls. Each gene was targeted by 5 or more independent shRNAs tested individually in a 384 well format using robotic manipulations. A total of 6,144 individual shRNAs were incubated with IM-9 myeloma cells and subsequently tested for their ability to modulate response by NKL effector cells (an IL-2 dependent human NK cell line). The top 10 percentile of shRNAs inducing increased secretion of interferon-gamma (INF-γ) from NKL cells was identified. To reduce the likelihood of off-target effects, only genes that were positive for 2 or more independent shRNAs were selected for further analysis. Among the genes that increased target cell susceptibility to NK activity we found 2 members of the Jak family (Jak1 and Jak2) with Jak1 being one of the strongest hits in our screen. IM-9 myeloma cells with stable expression of at least 2 independent shRNAs targeting Jak1 and Jak2 were established and tested for their sensitivity to NKL, NK92 or primary NK cells using INF-γrelease and 51Cr release cytotoxicity assays. Stable suppression of both Jak1 and Jak2 in IM-9 cells induced a significant increase of INF-γsecretion from NK cells and increased sensitivity in cytotoxicity assay when compared to parental IM-9 or cells expressing irrelevant shRNAs. Western blot analysis showed a selective decrease of Jak1 and Jak2 protein in IM-9 cells expressing specific shRNAs but not irrelevant shRNAs. While target cells with reduced expression of Jak1 and Jak2 were more susceptible to NK cell activity, no effect was observed when Jak3 and TYK2 were silenced. We then tested the NK susceptibility of different tumor cell lines with reduced expression of Jak1 and Jak2. Seven additional tumor cell lines representing other hematologic malignancies expressing Jak1 and Jak2 shRNAs were established: myeloma (KM12BM), chronic myeloid leukemia (K562), Burkitt's lymphoma (Daudi), acute myeloid leukemia (U937, ML2 and KG1) and acute T cell leukemia (Jurkat). These experiments confirmed that Jak1 silencing can induce increased susceptibility to NK cell activity. However, this effect was more pronounced in some cases (IM-9, KM12BM, U937, KG1) compared to other cell lines where this effect was limited (K562, ML-2, Jurkat) or absent (Daudi). To investigate the mechanism for modulation of target cell susceptibility to NK cells by Jak1, we compared gene expression profiles of IM-9-Jak1-KO with IM-9 parental cells and IM-9 cells infected with an irrelevant shRNA. No difference in expression was found for ligands of activating NKG2D receptors (MICA, MICB, ULPB1, 2, 3) or ligands for NK inhibitory receptors (HLA Class I genes A, B, C, E). One of the most up-regulated genes in IM-9-Jak1-KO cells was TNFSFR10A (TRAIL-R1), a gene that is known to induce apoptotic signals upon TRAIL engagement. In contrast, FAIM3, an inhibitor of FAS (CD95) signaling, was significantly down-regulated. IM-9-Jak1-KO cells also over-expressed several GALNT genes, recently shown to be markers of TRAIL sensitivity. These results suggest that Jak1 and possibly Jak2 can modulate susceptibility of some tumor cells to NK cell lysis. The mechanism for this effect appears to be at least partly through increased sensitivity to engagement of the TRAIL/FAS extrinsic apoptotic pathway. Disclosures:No relevant conflicts of interest to declare.

Janus kinase inhibitor INCB20 has antiproliferative and apoptotic effects on human myeloma cellsin vitroandin vivo

Protein tyrosine kinases of the Janus kinase (JAK) family are associated with many cytokine receptors, which, on ligand binding, regulate important cellular functions such as proliferation, survival, and differentiation. In multiple myeloma, JAKs may be persistently activated due to a constant stimulation by interleukin (IL)-6, which is produced in the bone marrow environment. INCB20 is a synthetic molecule that potently inhibits all members of the JAK family with a 100- to 1,000-fold selectivity for JAKs over >70 other kinases. Treatment of multiple myeloma cell lines and patient tumor cells with INCB20 resulted in a significant and dose-dependent inhibition of spontaneous as well as IL-6-induced cell growth. Importantly, multiple myeloma cell growth was inhibited in the presence of bone marrow stromal cells. The IL-6 dependent cell line INA-6 was particularly sensitive to the drug (IC50<1 micromol/L). Growth suppression of INA-6 correlated with an increase in the percentage of apoptotic cells and inhibition of signal transducer and activator of transcription 3 phosphorylation. INCB20 also abrogated the protective effect of IL-6 against dexamethasone by blocking phosphorylation of SHP-2 and AKT. In contrast, AKT phosphorylation induced by insulin-like growth factor-I remained unchanged, showing selectivity of the compound. In a s.c. severe combined immunodeficient mouse model with INA-6, INCB20 significantly delayed INA-6 tumor growth. Our studies show that disruption of JAKs and downstream signaling pathways may both inhibit multiple myeloma cell growth and survival and overcome cytokine-mediated drug resistance, thereby providing the preclinical rationale for the use of JAK inhibitors as a novel therapeutic approach in multiple myeloma.

Proteomic Analysis for the Identification of Disease Biomarkers and Therapeutic Targets in Natural Killer Cell Lymphoma.

Natural killer cell lymphoma (NKL) is a highly aggressive form of hematolymphoid malignancy that arises from natural killer cells, a critical component of the innate immune system. Early diagnosis of the tumor is critical however there are no biomarkers which are useful currently for the diagnosis and monitoring of patients with NKLs. We hypothesized that comprehensive analysis of differentially expressed proteins by NKL and normal NK cells would be a rational strategy for the discovery of NKL biomarkers and lead to better understanding of pathogenetic mechanisms of NKL. We used total cell lysates obtained from highly enriched peripheral blood NK cells (CD3-, CD56dim) and an Epstein-Barr virus negative, IL-2 dependent cell line (NK-92MI) for quantitative proteomic analysis. Isotopecoded affinity tags (ICATTM)-labeling followed by liquid chromatography-tandem mass spectrometry analyses (LC-MS/MS) identified 114 differentially expressed proteins in NKL compared to the normal NK cells. Out of these, 80 were upregulated 1.5-fold or greater and 34 were downregulated 1.5-fold or greater in NKL. Proteins within diverse cellular functional categories including cell signaling, transcriptional regulation, DNA metabolism, and immune response were differentially expression. Furthermore, known proteins relevant to the function of NK cells such as perforin, granzyme and defensins were downregulated in the NKL cell line. We selected eight proteins to validate the changes identified by ICAT-LC-MS/MS using Western blot analysis. HSP90, PCNA, alpha-enolase, GSTP1, galectin-1, and PARK7 were upregulated, and lactoferrin and annexin A1 were downregulated by western blot analysis demonstrating high concordance with the results of ICAT-LC-MS/MS analysis. We validated the functional significance of HSP90 as a potential therapeutic target in NKL by investigating the effect of inhibitors of HSP90 (geldanamycin) on the survival of an NKL cell line. Inhibition of HSP90 led to significant decrease in cell viability and apoptotic cell death as confirmed by western blotting demonstration of PARP-1 induction and detection of its cleavage products. This study represents the first global quantitative proteomic cataloguing of proteins expressed by NKL and normal NK cells, and reveals potential candidate proteins that may be useful for the diagnosis and function as therapeutic targets in patients with NKL.

The Immunotoxin HM1.24-ETA′ Is a Potent Inducer of Apoptosis in Malignant Plasma Cells

Despite new treatment modalities, the clinical outcome of at least a subgroup of patients with multiple myeloma (MM) still needs improvement. Antibody-based targeted therapies are increasingly used for tumor therapy, and may represent interesting options for MM patients. HM1.24 is a surface molecule that is over expressed on malignant plasma cells and efficiently internalized from the cell surface. It may represent a promising target for the development of myeloma-directed immunoconstructs. Here, the development and characterization of a novel single-chain immunotoxin, HM1.24-ETA′, is described. HM1.24-ETA′ was generated by genetic fusion of an HM1.24-specific single-chain Fv (scFv) antibody and a truncated variant of Pseudomonas aeruginosa exotoxin A (ETA′). The immunotoxin was expressed in E. coli and purified to homogeneity by affinity chromatography. Specific binding to HM1.24 was demonstrated by immunofluorescence staining and flow cytometry using antigen positive and negative cells. HM1.24-ETA′ efficiently inhibited growth of IL-6 dependent and IL-6 independent myeloma cell lines (INA-6, RPMI8226, U266). Half maximal growth inhibition was observed at low nanomolar concentrations. Further analyses demonstrated that target cell killing occurred via induction of apoptosis, as evidenced by Annexin V/propidium iodide staining and detection of PARP cleavage. The cytotoxic effect was completely blocked by adding excess of unconjugated parental antibody, demonstrating that the effect was antigen-specific and not mediated by non-specific uptake of the immunotoxin. Importantly, HM1.24-ETA′ efficiently triggered apoptosis (>80% Annexin V positive cells) of freshly isolated plasma cell leukemia cells within 48h. In conclusion, HM1.24-ETA′ efficiently triggered apoptosis of multiple myeloma cell lines as well as freshly isolated tumor cells. These results indicate that HM1.24 may represent a promising target structure for efficient antigen-specific delivery of cytotoxic compounds to plasma cell tumors.

Characterization of the Effects of Pim Kinase Inhibition on Multiple Oncogene-Driven Cell Lines

We report here the biological effects of inhibiting Pim-1, -2 and -3 serine/threonine protein kinases in cells and in vivo using a novel, potent and selective small molecule inhibitor directed against these kinases. In vitro enzyme assays revealed potent inhibition of all three Pim kinase isoforms while maintaining selectivity against more than 220 other protein kinases. In cells, compound treatment produced biological effects consistent with Pim inhibition, including a reduction in BAD protein phosphorylation. Moreover, this biological activity corresponds well with the compound's anti-proliferative and pro-apoptotic activity in the IL-3 dependent mouse pro-B cell line Ba/F3 and in factor-indpendent Ba/F3 cell lines dependent upon the expression of BCR-Abl or TEL-JAK2 for growth and survival. Importantly, compared to the BCR-Abl driven Ba/F3 cell line, this compound was equally effective at blocking the growth of an Imatinib-resistant BCR-Abl[ T315I]-driven Ba/F3 cell line. Finally, this compound possesses pharmacokinetic properties that predict the potential for in vivo activity. Therefore, we assessed the effect of Pim kinase inhibition on tumor growth in a mouse subcutaneous tumor xenograft model employing a cell line driven by the TEL-JAK2 oncogene. Results demonstrated that the compound significantly inhibited tumor growth in a dose-dependent manner. In terms of potential clinical utility, these data show that Pim kinase inhibition could be an effective therapeutic strategy for numerous hematologic malignancies including chronic and acute myelogenous leukemias, particularly in those patients that have become resistant to existing therapies such as Imatinib.

Comparative Analysis of LFA-1 Activation State and Functional Involvement in Enhanced Cytotoxicity of NK Cell Lines KHYG-1 and NK-92

Background: KHYG-1 and NK-92 are highly cytotoxic IL-2 dependent NK cell lines. NK cytotoxicity is regulated through an array of receptors with activating and inhibitory functions for spontaneous elimination of pathogen infected or tumor cells. Important roles have been described for the leukocyte function-associated antigen (LFA)- 1 adhesion receptor (αLβ2) in NK cytotoxicity. In T cells, the integrin LFA-1 occurs in a non-functional, bent form and requires activation, through divalent cations e.g., for ligand binding. In marked contrast, IL-2 stimulated NK cells could directly engage the LFA-1 ligand intercellular adhesion molecules (ICAM)-1, thereby inducing conjugate formation, granule polarization, degranulation, and tumor cell lysis (Barber DF et al., 2004. JI 173:3653–9). Strikingly, in the NK cell line KHYG-1 granules were constitutively polarized (clustered around the MTOC; Suck G et al., 2006. Int Immunol 18:1347–54) and LFA-1 downstream signaling molecules, the spleen tyrosine kinase (Syk) and extracellular signal-regulated MAP kinase (ERK) constitutively phosphorylated (Suck G et al., 2005. Exp Hem. 33:1160–71). These previous findings prompted us to investigate LFA-1 activation state and functional involvement in cytotoxicity in KHYG-1 and NK- 92.Methods: Adhesion assays were performed on immobilized ICAMs and % adherence determined in a fluorescence plate reader (Tang XY et al., 2008 JI, 180:4793–804). LFA-1 was activated with Mg/EGTA (5 mM MgCl2 and 1.5 mM EGTA), monoclonal antibody (mAb) KIM185 (10 mg/mL) or both; LFA-1 mAb, clone MHM24, was used for blocking studies, 1–10 mg/ml and mAb KIM127 for immunoprecipitation; cytotoxicity, conjugate formation, and degranulation (CD107a) were measured by Flow cytometry and cell morphology imaged by phase contrast (Olympus, 1X70) or confocal microscopy (Zeiss, LSM510 Meta/Nikon A1). Excel software was employed for statistical analyses.Results: In cell binding assays KHYG-1 and NK-92 showed constitutive adhesion to the LFA1-1 ligand ICAM-1, 61% +/− 5.8% SD and 55% +/− 7.5% SD, respectively. However, only 22% NK-92 and 10.5% KHYG-1 cells adhered to the lower affinity ligand ICAM-3 and activation with Mg/EGTA or KIM185 was required to increase binding to 73% in KHYG- 1 and to 62% in NK-92. Immunoprecipitation experiments with the activation reporter mAb KIM127 revealed an activated extended LFA-1 conformation in both cell lines. Together these results suggested an intermediate affinity activation state for the LFA- 1 population in KHYG-1 and NK-92. Despite such similarities, ICAM-1 engagement triggered pronounced cell spreading in KHYG-1, similar to phorbol ester stimulated T cells, but not in NK-92 or primary NK cells. It is conceivable that the capacity to undergo cytoskeletal remodeling in KHYG-1 may be impaired as potentially also reflected in the constitutively polarized granule state. In addition, LFA-1 blocking studies with LFA- 1 antibody MHM24 did not inhibit target conjugate formation in KHYG-1, compared to partial inhibition in NK-92, and cytotoxicity against K562 was only about 40% diminished in KHYG-1, compared to almost complete abrogation (maximum 98%) in NK-92. Nevertheless, overall cytolytic potential against K562 was comparable among the two cell lines, which implicated important functions for other receptors at least in KHYG-1.Conclusion: Results suggested a constitutively activated intermediate affinity state for LFA-1 in KHYG-1 and NK-92. It is conceivable that a similar activated state occurs in primary NK cells. Interestingly, although cytoskeletal dysfunction was indicated in KHYG-1, cytotoxicity of the cell line was unaffected, rendering it a valuable model for the study of alternative pathways.

A Random shRNA-Encoding Library for Phenotypic Selection and Hit-Optimization

RNA interference (RNAi) is a mechanism for inhibiting gene expression through the action of small, non-coding RNAs. Most existing RNAi libraries target single genes through canonical pathways. Endogenous microRNAs (miRNAs), however, often target multiple genes and can act through non-canonical pathways, including pathways that activate gene expression. To interrogate all possible functions, we designed, synthesized, and validated the first shRNA-encoding library that is completely random at the nucleotide level. Screening in an IL3-dependent cell line, FL5.12, yielded shRNA-encoding sequences that double cell survival upon IL3 withdrawal. Using random mutagenesis and re-screening under more stringent IL3-starvation conditions, we hit-optimized one of the sequences; a specific nucleotide change and the creation of a mismatch between the two halves of the stem both contributed to the improved potency. Our library allows unbiased selection and optimization of shRNA-encoding sequences that confer phenotypes of interest, and could be used for the development of therapeutics and tools in many fields of biology.

Apoptotic Cell-Mediated Immunoregulation of Dendritic Cells Does Not Require iC3b Opsonization

A number of recent studies show that activation of CR3 on dendritic cells (DCs) suppresses TLR-induced TNF-alpha and IL-12 production and inhibits effective Ag presentation. Although the proposed physiologic role for these phenomena is immune suppression due to recognition of iC3b opsonized apoptotic cells by CR3, all of the aforementioned investigations used artificial means of activating CR3. We investigated whether iC3b opsonized apoptotic cells could induce the same changes reported with artificial ligands such as mAbs or iC3b-opsonized RBC. We explored the kinetics of iC3b opsonization in two models of murine cell apoptosis, gamma-irradiated thymocytes and cytokine deprivation of the IL-3 dependent cell line BaF3. Using a relatively homogenous population of early apoptotic cells (IL-3 deprived BaF3 cells), we show that iC3b opsonized apoptotic cells engage CR3, but this interaction is dispensable in mediating the anti-inflammatory effects of apoptotic cells. TLR-induced TNF-alpha and IL-12 production by bone marrow-derived DCs occurs heterogeneously, with apoptotic cells inhibiting only certain populations depending on the TLR agonist. In contrast, although apoptotic cells induced homogeneous IL-10 production by DCs, IL-10 was not necessary for the inhibition of TNF-alpha and IL-12. Furthermore, because the ability of iC3b opsonization to enhance phagocytosis of apoptotic cells has been controversial, we report that iC3b opsonization does not significantly affect apoptotic cell ingestion by DCs. We conclude that the apoptotic cell receptor system on DCs is sufficiently redundant such that the absence of CR3 engagement does not significantly affect the normal anti-inflammatory processing of apoptotic cells.

Increased spontaneous secretion of IL-6 from B cells of patients with B chronic lymphatic leukaemia (B-CLL) and autoimmunity

We studied B cells from 18 patients with B-CLL, six of them with autoimmune haemolytic anaemia, for spontaneous secretion of IL-6. Our aim was to determine whether the increased incidence of autoimmune disease found in B-CLL patients is associated with enhanced spontaneous IL-6 secretion. IL-6 was measured by the effect of B cell supernatants on the proliferation of an IL-6 dependent plasmacytoma cell line T1165. The highest IL-6 values (7.4 +/- 1.8 U/ml) were measured in supernatants derived on day 3 of culture from lymphocytes of the six patients with B-CLL and concomitant autoimmune disease. The maximal IL-6 values for 10 patients with B-CLL only were 2.8 +/- 0.3 U/ml and for 10 age-matched controls, 0.8 +/- 0.3 U/ml (P less than 0.01, each group compared with the other). We conclude that there is an association between B-CLL, autoimmune disease and the spontaneous in vitro secretion of IL-6. Further studies are needed to determine whether the IL-6 secretion plays a role in the pathogenesis of autoimmune disease in patients with B-CLL.

IL-2– and IL-15–induced activation of the rapamycin-sensitive mTORC1 pathway in malignant CD4+ T lymphocytes

We examined functional status, activation mechanisms, and biologic role of the mTORC1 signaling pathway in malignant CD4(+) T cells derived from the cutaneous T-cell lymphoma (CTCL). Whereas the spontaneously growing CTCL-derived cell lines displayed persistent activation of the TORC1 as well as the PI3K/Akt and MEK/ERK pathways, the IL-2-dependent cell lines activated the pathways in response to IL-2 and IL-15 but not IL-21. Activation of mTORC1 and MEK/ERK was nutrient dependent. The mTORC1, PI3K/Akt, and MEK/ERK pathways could also be activated by IL-2 in the primary leukemic, mitogen-preactivated CTCL cells. mTORC1 activation was also detected in the CTCL tissues in the lymphoma stage-dependent manner with the highest percentage of positive cells present in the cases with a large cell transformation. Rapamycin inhibited mTORC1 signaling and suppressed CTCL cell proliferation but showed little effect on their apoptotic rate when used as a single agent. Activation of the mTORC1, PI3K/Akt, and MEK/ERK pathways was strictly dependent on the Jak3 and Jak1 kinases. Finally, mTORC1 activation was transduced preferentially through the PI3K/Akt pathway. These findings document the selective gammac-signaling cytokine-mediated activation of the mTORC1 pathway in the CTCL cells and suggest that the pathway represents a therapeutic target in CTCL and, possibly, other T-cell lymphomas.

Evaluation of XL999, a Potent Inhibitor of FGFR3, for the Potential Treatment of t(4;14) Positive Multiple Myeloma.

Recent advances in our understanding of the molecular pathogenesis of Multiple Myeloma (MM) have provided novel therapeutic targets for treatment of this incurable malignancy. The association of Fibroblast Growth Factor Receptor 3 (FGFR3) with the t(4;14) translocation that occurs uniquely in approximately 15% of MM patients, coupled with the demonstration of the transforming potential of this receptor tyrosine kinase (RTK), make this a particularly attractive target for the development of therapeutic agents for this poor prognosis group. Indeed, inhibition of activated FGFR3 induces apoptosis and inhibits growth in mice of FGFR3-expressing MM tumors, providing further validation of this RTK as a therapeutic target in t(4;14) MM. XL999 is a novel small molecule inhibitor of kinases involved in tumor cell growth and angiogenesis, and is currently in a Phase 1 clinical trial in patients with non-small cell lung cancer. XL999 inhibits members of the class III-V RTKs, including FGFR1 and 3, VEGFR2, PDGFR? and ?, KIT, and FLT3, and exhibits pharmacodynamic target modulation, anti-angiogenesis, and efficacy in multiple tumor models in mice (Eur. J. Cancer Suppl. 2: 141). We report here the FGFR3-targeting characteristics and anti-myeloma activity of XL999. XL999 inhibits FGFR3 with low nanomolar potency in biochemical enzyme assays. Therefore, we examined the activity of XL999 in several cell-based mechanistic assays of FGFR3 activity. The IL-6 dependent cell line, B9, was engineered to express wild-type or activated mutants (Y373C, K650E, G384D and J807C) of FGFR3 found in MM. XL999 differentially inhibited FGF-mediated growth of B9 cells expressing wild-type and mutant receptors, with IC50 values in the low nM range. Growth of these cells could be rescued by IL-6, indicating selectivity of XL999 for FGFR3. The activity of XL999 against FGFR3 was also examined in t(4;14) positive MM cell lines expressing FGFR3. XL999 inhibited the proliferation and viability of KMS-11 cells (FGFR3-Y373C) and OPM-2 cells (expressing FGFR3-K650E) with low nM potency. Importantly, inhibition by XL999 was still observed in the presence of IL-6 or IGF1, potent growth factors for MM cells. In addition, XL999 potently inhibited the phosphorylation of FGFR3 in OPM-2 and KMS-11 tumors after a single oral dose in pharmacodynamic studies in nude mice. U266 and 8226 cells, which lack FGFR3 expression, displayed minimal growth inhibition by XL999, demonstrating that at effective concentrations, XL999 is selective and exhibits minimal nonspecific cytotoxicity to FGFR3 negative MM cells. Further characterization of this finding demonstrated that inhibition of cell growth corresponded to G0/G1 cell cycle arrest, dose-dependent inhibition of downstream ERK phosphorylation, and induction of apoptosis in FGFR3-positive cells. In vitro analysis of XL999 combined with melphalan, bortezomib, or dexamethasone applied simultaneously to KMS11 cells indicated a strong synergistic interaction with dexamethasone, and additivity with either melphalan or bortezomib. Finally, XL999 produced cytotoxic responses in 5/5 primary myeloma samples derived from patients harboring a t(4;14) translocation. Collectively, these results suggest that XL999 may hold potential for patients with MM, particularly in combination with other agents.

Leukemic Transformation in Mice Expressing a NUP98-HOXD13 Transgene Is Accompanied by Spontaneous Mutations in Receptor Tyrosine Kinase Pathway Genes.

The NUP98-HOXD13 (NHD13) fusion gene, formed by the t(2;11)(q31;p15), has been identified in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). We recently reported that mice which express an NHD13 transgene develop MDS; over half of these mice eventually develop acute leukemia between six and fourteen months. An emerging paradigm holds that at least one event from each of two classes is required for leukemic transformation: Class I (proliferation or survival promoting events) and Class II (differentiation blocking events). The NHD13 fusion protein has previously been shown to fit the criteria of a Class II event, as it impairs differentiation of haematopoietic cells. Therefore, we used a candidate gene approach that focused on Class I mutations to search for secondary genetic events that might collaborate with the NHD13 transgene. Since the most frequently mutated Class I genes in human myeloid leukemia are FLT3 (24%), NRAS (16%), KIT (5%) and KRAS (4%), we sequenced relevant regions of these genes (Flt3, exons 14, 15 and 20; Kit, exons 8 and 17; Nras, exons 2 and 3; and Kras, exons 2 and 3) in a cohort of 26 mice with AML. We found no Flt3 or Kit mutations in this cohort; however, we identified 3 (12%) Nras and 3 (12%) Kras mutations. All six of the ras mutations identified in the AML cohort occurred within codon 12, which is predicted to result in a constitutively active ras protein. We also screened a smaller sample set of NHD13 mice that developed pre-T lymphoblastic leukemia/lymphoma and found 0/8 Nras mutations but 1/8 (13%) Kras mutation; this mutation occurred in codon 13. CBL is a ubiquitin ligase that is responsible for targeting degradation of selected receptor tyrosine kinases. Intriguingly, CBL mutations that lead to FLT3 activation have recently been identified in AML patients. Therefore, we screened the NHD13 AML cohort for mutations in Cbl and identified a deletion of 297 bp that results in loss of the exon 8 splice acceptor site. RT-PCR demonstrated an aberrant Cbl cDNA which lacks the RING finger domain encoded by exon 8. We have previously reported the derivation of an IL-3 dependent haematopoietic cell line from embryonic stem (ES) cells that expressed an NHD13 “knock-in” allele. Preliminary results indicate that the activated ras mutations are capable of transforming the cell line to IL-3 independence, supporting the contention that the ras mutations (Class I) complement the NHD13 fusion protein (Class II). We are currently investigating the potential of the Cbl mutant to transform the IL3-dependent cell line. Although the current paradigm for AML predicts these results, this is the first report of spontaneous Class I (Cbl or ras) mutations that complement a defined, genetically engineered Class II mutation in an animal model of AML. These results have general implications for other oncogenic mouse models, as well as specific implications for the nature of NHD13-mediated leukemogenesis.

Overexpression of Rheb2 Has Diverse Effects on Hematopoietic Progenitor and Stem Cell Functions.

Determining which intracellular signaling pathways are important for hematopoietic stem and progenitor cell functions is important for therapeutic uses of these cells. The intracellular serine/threonine kinase mammalian target of rapamycin (mTOR) is important for regulation of the growth, proliferation, survival and differentiation of various cell types in the body, including normal hematopoietic and blood cancer cells. We chose to study the protein Ras homologue enriched in brain-2 (Rheb2), a known activator of mTOR signaling, because there is evidence it is preferentially expressed in highly purified mouse (m) hematopoietic stem cell populations (Ivanova et al. Science , 298(5593):601–04, 2002). Rheb2 is one of two members of the Rheb small GTPase family of proteins known to activate mTOR by mechanisms which are not fully elucidated, but that are regulated by Akt and the tuberous sclerosis complex (TSC1/2). We overexpressed mouse Rheb2 protein in both primary mouse bone marrow (mBM) cells and two mouse hematopoietic cell lines (BaF3 and 32D) and determined effects on progenitor cell colony-formation, delayed IL-3 driven proliferation in cell lines and hematopoietic stem cell function in an in vivo competitive stem cell repopulation assay. Overexpression of Rheb2 in retrovirally transduced mBM cells significantly increased the colony-forming ability of the cells in response to growth factors. The positive effect of Rheb2 was most pronounced on granulocyte-macrophage (CFU-GM) progenitors (greater than two-fold increase over empty vector transduced cells) and was persistent in cells expanded in liquid culture up to one week after FACS sorting of transduced cells. Additionally, Rheb2 overexpression significantly increased the ability of mBM cells to form CFU-GM colonies under conditions of delayed-growth factor addition (at 24 and 48 hours after plating), suggesting a positive role for Rheb2 in the survival of these cells. Interestingly, addition of the pro-survival chemokine SDF-1/CXCL12 to the delayed-growth factor colony assay plates enhanced the survival of the empty vector transduced cells but did not further enhance the survival of the Rheb2 overexpressing cells. Using two IL-3 dependent hematopoietic cell lines, BaF3 and 32D, to further assess Rheb2 function and signaling, we found overexpression of Rheb2 enhanced proliferation after delayed addition of IL-3. These changes correlated with an increase in mTOR signaling in Rheb2 transduced cells. In order to evaluate the effect of Rheb2 overexpression on hematopoietic stem cell function, an in vivo competitive repopulation assay was performed. Transduced donor and non-transduced competitor cells were pooled at two ratios (1:1 and 0.4:1 donor to competitor) and injected into lethally irradiated recipients. Transduced cells were followed in the recipients by measuring GFP+ cell chimerism. In contrast to the data on progenitor cell function, transduced Rheb2 cells had significantly decreased competitive repopulation in the recipient mice (greater that 3-fold decrease compared to empty vector). This suggests that Rheb2 overexpression may negatively affect HSC homing, stable engraftment and/or self-renewal. Overall, these studies demonstrate that overexpression of Rheb2 positively enhances progenitor cell functions such as colony-formation, while negatively affecting stem cell competitive repopulation, thereby providing a possible regulatory mechanistic link between the flow from the stem to progenitor cell compartment.

Effect of the green tea polyphenol epigallocatechin gallate (EGCG) on growth and IL-6 signalling pathways in malignant plasma cells

8114 Background: Epigallocatechin gallate (EGCG) is the predominant polyphenolic constituent of green tea leaves that possesses antitumor, antiinflammatory, and antioxidant activity. EGCG exerts its effects through potentially multiple mechanisms including inhibition of growth factor receptor signalling. The compound is currently under investigation in a phase I/II clinical trial for treatment of patients with early stage chronic lymphocytic leukemia at Mayo Clinic. The goal of our study was to examine the in vitro effects of EGCG in multiple myeloma (MM). Methods: A panel of human myeloma cell lines (n=6) including the IL-6 dependent INA-6 cell line was used to evaluate the sensitivity to EGCG. Cells were cultured for three days in the absence or presence of EGCG at concentrations between 6.25 μM and 100 μM. Cell viability was determined in a colorimetric tetrazolium (MTS) based assay and by trypanblue exclusion. For signalling experiments, INA-6 cells were IL-6 and serum starved and then treated with EGCG for two hours before IL-6 was added. Whole cell lysates were prepared and subjected to SDS-PAGE and Western blot analysis. Results: EGCG inhibited the in vitro growth of human myeloma cell lines by inducing cell death in a time and dose-dependent manner. IC50 concentrations were between 12,5 μM and 50 μM. IL-6 mediated growth of INA-6 cells was inhibited at similar doses. The addition of excess amounts of IL-6 could not protect from EGCG induced cytotoxicity. Pretreatment of INA-6 cells with EGCG resulted in a dose-dependent inhibition of IL-6 induced STAT3 tyrosine phosphorylation. In these cells, stimulation with IL-6 leads to upregulation of Mcl-1 expression. In contrast, phosphorylation of p44/p42 MAPK, which is constitutively activated in INA-6 cells, was not affected. Conclusion: EGCG has growth inhibitory activity on myeloma cells. Specific inhibition of signalling pathways that regulate expression of anti-apoptotic proteins could be one mechanism how EGCG exerts its activity. Our work provides the rationale for further studies to evaluate the effect of EGCG not only in B-CLL, but also in plasma cell tumors. No significant financial relationships to disclose.

NK Cell Mediated Tumor Cytotoxicity is enhanced by Mild Thermal Stress through Heat Shock Factor 1 (50.24)

Abstract Natural killer (NK) cells directly kill tumor cells and regulate downstream adaptive immune responses. Previous studies in our lab suggest that mild thermal stress (39.5 C) significantly enhances the cytotoxicity of human peripheral blood NK cells compared to normothermic conditions (37 C) against human colon tumor cells. This enhancement was not seen when syngeneic human PBMC were used as targets, nor when the IL-2 dependent human NK cell lines NKL or NK92MI, or IL-2 activated primary NK cells were used as effectors. Thus, we hypothesized that resting NK cell cytotoxic activity is regulated by mild thermal stress. Blocking of the activating receptor NKG2D and its stress-induced ligand MICA with blocking antibodies suggested the involvement of these molecules in the thermal regulation of NK cytotoxicity. We found that inhibitory receptor KIR2DL1 levels on NK cells were not changed with thermal stress. Increased clustering of NKG2D on NK cells was observed with thermal stress, suggesting the potential for enhanced efficiency of activating signals. However prevention of lipid raft clustering with methyl B-cyclodextrin on NK cells shows no significant affect on thermally enhanced cytotoxicity. On target cells, MICA expression appeared to be upregulated by mild thermal stress in a manner that is dependent on the transcription factor HSF-1. Overall, these data suggest that thermal therapy might be clinically utilized to help enhance NK mediated tumor cell killing. Supported by NIH P01 CA94045, R01 CA71599, Komen Foundation DISS0402487 and Fulbright Program.

Possible Roles of KIR2DL4 Expression on uNK Cells in Human Pregnancy

To investigate possible roles of the natural killer (NK) cell receptor killer immunoglobulin-like receptor (KIR)2DL4 expressed on uterine NK (uNK) cells during pregnancy, we investigated KIR2DL4 expression on uNK cells isolated from patients with early recurrent spontaneous abortion (RSA) and normal early pregnancy women, and functions of KIR2DL4 was analyzed in vitro. METHODS OF THE STUDY: Semi-quantitative RT-PCR analysis was introduced to detect KIR2DL4 messenger RNA (mRNA) expression on uNK cells. Cytotoxicity and cytokine production as the result of interaction of KIR2DL4 and its ligand human leukocyte antigen (HLA)-G were analyzed in vitro with lactic dehydrogenase releasing method and enzyme-linked immunosorbent assay, respectively. No significant difference in KIR2DL4 mRNA expression was observed, while the KIR2DL4 protein level in isolated uNK cells is much higher in normal controls than that in RSA patients. Data showed that HLA-G transfection could not reverse the lysis of uNK against HLA-G transfected K562 cells but induced cytokine production. Furthermore, we demonstrated that, via KIR2DL4, membrane-bound HLA-G could induce high cytotoxicity and cytokine production in a high cytotoxic, IL-2 dependent human NK cell line NK-92 cells. Our data suggest that KIR2DL4 might play a crucial implication for human pregnancy.

Induction of Constitutive High-Level Expression of c-Myc in 32D Cells by Mycoplasmas is Associated with Their Ability to Prevent Apoptosis and Induce Malignant Transformation

Our previous studies showed that infections by mycoplasmas could prevent apoptosis and induce the malignant transformation of mammalian cells. Other studies indicate that c-Myc plays an important role in promoting apoptosis and malignant transformation of cells. To understand the role of c-Myc in the mycoplasma-induced apoptosis prevention and malignant cell transformation, 32D cells, an IL-3 dependent cell line, were infected and transformed by different species of mycoplasmas. The expression of Myc and ras gene families, apoptosis and the cell cycle during the infection and transformation were examined. Results showed that c-Myc expression was significantly increased in mycoplasma-transformed 32D cells. Withdrawal of IL-3 substantially decreased c-Myc expression in non-infected 32D cells and led to cell cycle arrest at the G1 phase followed by rapid apoptosis. Infection by M. fermentans or M. penetrans not only alleviated the sharp decrease of c-Myc expression, rescued 32D cells from cell-cycle arrest and prevented apoptosis in IL-3-free culture, but also induced autonomous growth of 32D cells. Although M. hominis and M. salivarium had the ability neither to prevent apoptosis nor to induce malignant transformation, they were still able to rescue the cells from cell cycle arrest at the G1 phase. The expression of ras family did not change significantly during the infection and transformation. These results suggest that constitutive expression of c-Myc appears to be associated with the continuous growth and malignant transformation of 32D cells induced by M. fermentans and M. penetrans, but not with rescuing the cell cycle arrest by the mycoplasmas.

Non‐viral gene delivery into primary natural killer lymphocytes

Goffinet and Keppler reported in The FASEB Journal on the use of nucleofection for gene delivery into primary rodent lymphocytes (1). Work from us (2) is cited as evidence that “lymphocyte populations other than T cells from humans and rodents have been considered virtually refractory to nonviral gene delivery.” This notion is misleading since in this (2), as well as in several other publications from our group (3, 4), it was clearly shown that nucleofection is an effective way to transfect human natural killer cells. The transfection efficiencies observed in Trompeter et al. (2) were more than 50% for the IL-2 dependent NK cell line NK3.3 as measured by surface expression of a H-2Kk reporter construct and about half the efficiency for primary NK cells as measured by luciferase activity. These numbers are actually in the same range as reported by Goffinet and Keppler for rodent NK cells. In addition, it was previously shown by us and others, that nucleofection is not only suitable for transfection of activated lymphocytes, which is recommended by Goffinet and Keppler, but is also efficient in transfection of resting NK and T cells in humans and rodents, respectively (2, 5). This observation makes nucleofection an attractive tool for immunotherapeutic use of “untouched” lymphocytes, as it enables non-viral gene delivery into lymphocytes without the need for polyclonal stimulation and thus unspecific activation.

Lyn Is an Important Component of the Signal Transduction Pathway Specific to FLT3/ITD and Can Be a Therapeutic Target in the Treatment of AML with FLT3/ITD.

Fms-like tyrosine kinase 3 (FLT3) is expressed in hematopoietic progenitor cells. An internal tandem duplication (ITD) of the FLT3 juxtamembrane domain (FLT3/ITD) is the most frequent mutation in human adult acute myeloid leukemia (AML). Being transfected into IL-3 dependent cell line, 32D, FLT3/ITD causes constitutive activation of FLT3 itself and its downstream signal components, which leads to IL-3 independent cell growth. In particular, STAT5 activation is thought to be a key signaling event since it is induced only by FLT3/ITD not by wild type FLT3. However the signal component which mediates FLT3/ITD to STAT5 is yet to be clarified. In the present study, we showed the mutant- specific association of FLT3/ITD with Lyn, which led to the phosphorylation of Lyn in vivo. We also demonstrated that Lyn directly bound to FLT3 through its SH2 domain in vitro and that the association depended on tyrosyl-phosphorylation at juxtamembrane domain of FLT3. We revealed the critical role of Lyn for STAT5 activation and the autonomous cell growth in FLT3/ITD transfectant of 32D by using anti-Lyn siRNA and the Src family kinase inhibitor PP2. More importantly, we demonstrated successful treatment of FLT3/ITD induced tumors with PP2 in the mice model. These results demonstrate that Lyn is a critical component of the signal transduction pathway specific to FLT3/ITD and can be a therapeutic target in the treatment of AML with FLT3/ITD.

Green Tea Polyphenol (−)-Epigallocatechin Gallate Inhibits IL-6 Mediated Signalling Pathways in Human Myeloma Cells.

Epigallocatechin-3-gallate (EGCG) is the predominant active ingredient of green tea leaves and has been shown to possess anti-tumor, anti-inflammatory, and anti-oxidant properties. EGCG confers its effects through potentially multiple mechanisms including inhibition of growth factor receptor signalling and decrease of cellular levels of anti-apoptotic proteins of the Bcl-2 family. We examined the effects of EGCG on a panel of human myeloma cell lines using EGCG concentrations ranging from 6.25–100 μM. After three days of culturing with EGCG, basal cell growth was inhibited in most of the cell lines as measured in an MTS based assay. IC50 concentrations were between 25 μM and 50 μM. IL-6 mediated growth of the IL-6 dependent INA-6 cell line was inhibited at similar doses. In these cells, stimulation with IL-6 leads to upregulation of Mcl-1 expression (Brocke-Heidrich et al., Blood. 2004; 103:242–251). Another green tea catechin, (−)-catechin gallate, seemed to be less active when used at identical concentrations. When INA-6 cells were pretreated for two hours with EGCG, a dose-dependent inhibition of IL-6 induced STAT3 tyrosine phosphorylation was observed as revealed by Western blot analysis. In contrast, phosphorylation of p44/p42 MAPK, which is constitutively activated in INA-6 cells, was not affected. The precise mechanism by which EGCG inhibits STAT3 phosphorylation remains to be determined. In conclusion, our results suggest to further evaluate the effect of EGCG not only in B-cell chronic lymphocytic leukemia, but also in plasma cell tumors.