The histone deacetylase inhibitor givinostat (ITF2357) exhibits potent anti-tumor activity against CRLF2-rearranged BCP-ALL.

Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult-to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.

Mass cytometry analysis reveals hyperactive NF Kappa B signaling in myelofibrosis and secondary acute myeloid leukemia.

Myeloproliferative neoplasms (MPNs) feature a malignant clone containing the JAK2 V617F mutation, or another mutation causing dysregulated JAK2 kinase activity. The multiple disease phenotypes of MPNs, and their tendency to transform phenotypically, suggest pathophysiologic heterogeneities beyond a common phenomenon of JAK2 hyperactivation. JAK2 has the potential to activate multiple other signaling molecules, either directly through downstream effectors, or indirectly through induction of target gene expression. We have interrogated myeloproliferative signaling in myelofibrosis (MF) and secondary acute myeloid leukemia (sAML) patient samples using mass cytometry, which allows the quantitative measurement of multiple signaling molecules simultaneously at the single-cell level, in cell populations representing a nearly complete spectrum of hematopoiesis. MF and sAML malignant cells demonstrated a high prevalence of hyperactivation of the JAK-STAT, MAP kinase, PI3 kinase and NFκB signaling pathways. Constitutive NFκB signaling was evident across MF and sAML patients. A supporting gene set enrichment analysis (GSEA) of MF showed many NFκB target genes to be expressed above normal levels in MF patient CD34+ cells. NFκB inhibition suppressed colony formation from MF CD34+ cells. This study indicates that NFκB signaling contributes to human myeloproliferative disease and is abnormally activated in MF and sAML.

Screening of retroviral cDNA libraries for factors involved in protein phosphorylation in signaling cascades.

We report a novel approach that allows for the rapid identification of proteins mediating phosphorylation in signaling cascades after specific stimulation. As a proof of concept, we used the interferon- gamma (IFN-gamma)-induced phosphorylation of signal transducer and activator of transcription-1 (Stat1) in a human promonocytic cell line, which was previously shown to be deficient in this signaling pathway. By using retroviral cDNA expression libraries, transduced selector cells expressing single cDNAs were stimulated with IFN-gamma, then fixed, permeabilized and stained intracellularly for phospho-Stat1 levels. Cells responding to the stimulation, which showed increased levels of phosphorylated Stat1, were enriched using fluorescence activated cell sorting (FACS). Genomic DNA was isolated from the enriched cell population and served as a template for cDNA amplification using PCR. After only one round of selection, a cDNA encoding the beta-chain of the IFN-gamma receptor (IFNGR2) was obtained and demonstrated to restore the selected phenotype. The approach now allows one to use phospho-events as reporters, alone or in tandem, for screening of signaling network states, overcoming a prior need to rely on the reporter genes that are often only indirect measures of phenotypes desired in a screen.

Oligonucleotide-directed site-specific integration of high complexity libraries into ssDNA templates.

We present an approach that generates an oligomer-based library with minimal need for restriction site modification of sequences in the target vector. The technique has the advantage that it can be applied for generating peptide aptamer libraries at sites within proteins without the need for introducing flanking enzyme sites. As an example we present a phagemid retroviral shuttle vector that can be used to achieve stable expression of the library in mammalian cells for the purpose of screening for peptides with desired biological activity.

Localized expression of an anti-TNF single-chain antibody prevents development of collagen-induced arthritis.

Although systemic administration of neutralizing anti-TNF antibodies has been used successfully in treating rheumatoid arthritis, there is a potential for side effects. We transduced a collagen reactive T-cell hybridoma with tissue-specific homing properties to assess therapeutic effects of local delivery to inflamed joints of anti-TNF single-chain antibodies (scFv) by adoptive cellular gene therapy. Cell culture medium conditioned with 1 x 10(6) scFv producer cells/ml had TNF neutralizing capacity in vitro equivalent to 50 ng/ml anti-TNF monoclonal antibody. Adding a kappa chain constant domain to the basic scFv (construct TN3-Ckappa) gave increased in vitro stability and in vivo therapeutic effect. TN3-Ckappa blocked development of collagen-induced arthritis in DBA/1LacJ mice for >60 days. Transgene expression was detected in the paws but not the spleen of treated animals for up to 55 days postinjection. No significant variations in cell proliferation or cytokine secretion were found in splenocytes or peripheral lymphocytes. IL-6 expression was blocked in the diseased paws of mice in the scFv treatment groups compared to controls. In conclusion, we have shown that local expression of an anti-inflammatory agent blocks disease development without causing demonstrable systemic immune function changes. This is encouraging for the potential development of safe adoptive cellular therapies to treat autoimmunity.

Induction of anti-tumor immunity in mice using a syngeneic endothelial cell vaccine.

Tumor endothelium could represent a novel target for active and passive immunotherapies of cancer. Here, we show that endothelial cells can be used as a vaccine in mice. In this study, three endothelial cell vaccine preparations from syngeneic (SVR), allogeneic (ISOS-1) and xenogeneic (ISO-HAS) sources were used to vaccinate mice. All mice developed humoral immune responses to endothelial cells and showed lower basal serum VEGF levels (37-45% lower) compared with unvaccinated control mice. Mice receiving the syngeneic SVR vaccine showed substantial inhibition of tumor growth after B16F10 melanoma challenge (50% of the mice in this group were tumor-free). The tumors that developed in the few mice in the syngeneic group had lower microvessel density counts (4-5 fold) compared with the other groups. The data suggests an in vivo antiangiogenic effect as the potential mechanism for the anti-cancer effect. In summary, further studies using other tumor models to demonstrate broad protection of this novel type of antiangiogenic vaccine are warranted.

Functional cloning of SPIN-2, a nuclear anti-apoptotic protein with roles in cell cycle progression.

The balance between hematopoietic cell viability and apoptosis is regulated by exogenous growth factors, however, the molecular mechanisms by which these trophic factors exert their effects remain obscure. A functional retroviral cDNA library-based screen was employed to identify genes that prevent growth factor withdrawal-mediated apoptosis in the myeloid progenitor cell 32Dcl3. This approach identified three classes of genes: those with known roles in apoptosis (bcl-X(L) and ornithine decarboxylase); genes previously identified but not linked directly to apoptotic signaling (O-linked N-acetylglucosamine transferase); and a previously uncharacterized gene we termed SPIN-2. In 32Dcl3 cells, expression of exogenous SPIN-2 provides 25% protection from apoptosis following growth factor withdrawal compared to controls which show approximately 1-2% survival. SPIN-2 overexpression slows cell growth rates and increases the percentage of cells in G(2)/M (32% vs control cells at 12%). Immunolocalization studies indicate that myc-epitope tagged SPIN-2 proteins, which retain their anti-apoptotic function, reside in the nucleus, whereas a C-terminal deletion mutant that loses its anti-apoptotic activity is located in the cytoplasm. These studies suggest that SPIN-2 is a novel nuclear protein that functions to regulate cell cycle progression and this activity is related to the inhibition of apoptosis following the removal of essential growth factors.

Resistance is futile: assimilation of cellular machinery by HIV-1.

HIV-1 budding appears to require Vps4 and Tsg101-two proteins that have links to endosomal sorting machinery. A picture emerges wherein divergent viruses recruit endosomal proteins like Tsg101 to gain access to ubiquitin processes that play a crucial role during viral budding.

A functional screen for genes inducing epidermal growth factor autonomy of human mammary epithelial cells confirms the role of amphiregulin.

To gain better understanding of the molecular alterations responsible for the aggressive growth potential of epidermal growth factor receptor (EGFR)-positive breast cancers, we utilized an expression cloning strategy to seek gene products that mediate the EGF-independent growth of human breast cancer cells. A retroviral cDNA expression library was constructed from the EGFR-positive SUM-149PT cell line, and transduced into growth factor-dependent human mammary epithelial (HME) cells. Recipient cells were functionally selected for their ability to proliferate in serum-free, EGF-free medium. Library cDNAs were recovered from EGF-independent colonies by PCR amplification or by biological rescue. Clone H55a#1 contained a library insert encoding amphiregulin. This EGFR ligand was able to confer EGF independence when transduced into HME cells. SUM-149PT and H55a#1 cells overexpressed amphiregulin transcripts, and secreted moderate EGF-like activity in conditioned media, indicating a possible autocrine loop. EGFR membrane levels and constitutive phosphorylation were consistent with this hypothesis, as well as the sensitivity of the cells to an ErbB-specific kinase inhibitor. Expression of the WT1 Wilms' tumor suppressor gene, a transcriptional activator of amphiregulin, did not parallel amphiregulin transcript levels, suggesting that another factor regulates amphiregulin in SUM-149PT. Our data confirm the importance of amphiregulin in the etiology of breast cancer.

Wnt signaling is required for thymocyte development and activates Tcf-1 mediated transcription.

T cell factor / lymphocyte enhancer factor (Tcf/Lef) transcription factors complex with the transcriptional co-activator beta-catenin to transduce Wnt signals in a variety of developmental systems. The prototypic family member Tcf-1 is highly expressed in T lineage cells. Tcf1-/- mice are defective in cell cycling of early thymocyte stages. Here, we show that the interaction of beta-catenin with Tcf-1 is required for full thymocyte development. This interaction may be established by signals mediated by Wnt1 and Wnt4, leading to increased Tcf-dependent transcriptional activity in thymocytes, as demonstrated in Tcf-LacZ reporter mice. Transduction of fetal thymocytes with Wnt1 and Wnt4 results in increased survival in an in vitro cell culture system. Retroviral expression of soluble Wnt receptor mutants that block Wnt signaling inhibits thymocyte development. These results imply an important role for the Wnt cascade in thymocyte development.

Combination angiostatin and endostatin gene transfer induces synergistic antiangiogenic activity in vitro and antitumor efficacy in leukemia and solid tumors in mice.

Angiostatin and endostatin are potent endothelial cell growth inhibitors that have been shown to inhibit angiogenesis in vivo and tumor growth in mice. However, tumor shrinkage requires chronic delivery of large doses of these proteins. Here we report synergistic antitumor activity and survival of animals when these factors are delivered in combination to tumors by retroviral gene transfer. We have demonstrated this efficacy in both murine leukemia and melanoma models. Complete loss of tumorigenicity was seen in 40% of the animals receiving tumors transduced by the combination of angiostatin and endostatin in the leukemia model. The synergy was also demonstrated in vitro on human umbilical vein endothelial cell differentiation and this antiangiogenic activity may suggest a mechanism for the antitumor activity in vivo. These findings imply separate pathways by which angiostatin and endostatin mediate their antiangiogenic effects. Together, these data suggest that a combination of antiangiogenic factors delivered by retroviral gene transfer may produce synergistic antitumor effects in both leukemia and solid tumors, thus avoiding long-term administration of recombinant proteins. The data also suggest that novel combinations of antiangiogenic factors delivered into tumors require further investigation as therapeutic modalities.

Retroviral transduction of a T cell receptor specific for an Epstein-Barr virus-encoded peptide.

The Type II EBV malignancies nasopharyngeal carcinoma and EBV(+) Hodgkin's disease express three subdominant antigens, latency membrane protein (LMP) 1, LMP2, and EBNA-1. While adoptive immunotherapy with T cell lines for Type III EBV malignancy (such as posttransplant lymphoma, PTLD, which expresses the immunodominant EBNA-3 antigens) has been used to prevent and treat PTLD, the generation of class I MHC-restricted CTL suitable for the immunotherapy of Type II EBV malignancy is difficult. This is primarily due to the lack of anti-LMP or EBNA-1 CTL activity in many healthy volunteers. We have engineered, by retroviral transduction of the TCR, CTL that have the potential to recognize subdominant EBV latency antigens. Using the SAMEN retroviral vector we demonstrate the ability to transfer CTL activity from a LMP2 peptide-specific CTL clone to a stimulated PBMC population. TCR-transduced PBMC also secrete IFN-gamma upon coculture with LMP2 targets and maintain expression of the transduced TCR during subsequent mitogenic expansion.

Dominant effector genetics in mammalian cells.

We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example-and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance-we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.

Statin-AE: a novel angiostatin-endostatin fusion protein with enhanced antiangiogenic and antitumor activity.

The combination of angiostatin and endostatin has been shown to have synergistic antiangiogenic and antitumor effects when the genes for these proteins are delivered to tumor cells by retroviral gene transfer. Here we report the construction of a murine angiostatin-endostatin fusion gene (Statin-AE) which shows enhanced antiangiogenic activity on human umbilical vein endothelial cell (HUVEC) tube formation in vitro compared with angiostatin or endostatin alone. Similarly, the fusion gene demonstrates antiangiogenic effects in vivo and antitumor activity in a B16F10 melanoma model when co-delivered by retroviral packaging cell inoculation in mice. The fusion gene demonstrates significantly greater inhibition of tumor growth compared with angiostatin, endostatin or the combination of genes.

Rapid production of retroviruses for efficient gene delivery to mammalian cells using 293T cell-based systems.

This unit details the applications of one of the more common retroviral packaging systems, based on the highly transfectable 293T cell. The packaging system employs the use of the Phoenix cell lines. Calcium phosphate-mediated transfection is described for efficient introduction of retroviral vector plasmid DNA into the cells to generate high yields of virion-containing supernatant. An alternate protocol describes a method for transfecting retroviruses that contain a vesicular stomatitis virus G (VSV G) protein. Such virions are said to be "pseudotyped" with VSV G glycoprotein. Support protocols provide a simple method for concentrating VSV-G-pseudotyped retroviruses, as well as methods for culturing, cryopreserving, thawing, and drug selecting the Phoenix packaging cell lines. Finally, several methods for transfecting adherent or suspension cells with retroviruses are described.

Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors.

Second- and third-generation three-plasmid vector systems, termed FELIX, were constructed from feline immunodeficiency virus (FIV). To enhance vector production, the weak FIV long terminal repeat promoter was replaced with the human cytomegalovirus enhancer/promoter. To construct a minimal system in which Gag-Pol was the only viral protein present, the cytoplasmic transport element was used in place of the FIV Rev-RRE system to facilitate nuclear export of Gag-Pol and the transfer vector. Unconcentrated vector titers routinely exceeded 1 x 10(6) IU/mL for most constructs tested. Second- and optimized third-generation vectors were capable of efficiently infecting G1/S- and G2/M-arrested cells. FIV-based FELIX vectors transduced human dendritic cells, hepatocytes, and aortic smooth muscle with efficiencies similar to that of a control 3T3 cell line. All three of these primary cell types were transducible by both the second- and third-generation FELIX vectors, demonstrating that FIV Gag-Pol alone contains the determinants necessary for transduction of primary cells. In cross-packaging tests, we observed that HIV Gag-Pol does not substantially package FIV vectors; consequently, use of such vectors in human immunodeficiency virus-infected cells should not lead to efficient mobilization of the inserted gene. Thus, this FIV-based vector system offers high efficiency and stable delivery of genes to numerous nondividing and primary cell types, opening new avenues for biological inquiry into normal human cells.

Retroviral delivery of peptide modulators of cellular functions.

Stable transduction of genetic material, in combination with sensitive methodologies for in vivo study of cell physiology, provides an opportunity to efficiently evaluate the functions of regulatory proteins. To dissect the minimal therapeutic function of such proteins, we have stably expressed protein microdomains as fusions, composed of short peptides, and detected specific subfunctions distinct from holoprotein function, using flow cytometry and other techniques. We demonstrate that retroviral delivery of the 24-amino-acid proliferating cell nuclear antigen-binding motif (p21C), derived from the C-terminus of the cell cycle inhibitor protein, p21, is sufficient to induce cell cycle arrest. Cells expressing this peptide motif reversibly execute both G1- and G2-checkpoint controls that are normally activated subsequent to interference with DNA synthesis. The p21C effect is distinct from results obtained with an intact p21 protein that also binds cyclin-CDK complexes and arrested cells exclusively at the G1/S transition. Thus, microdomains can exert unique biological effects compared to the parental molecules from which they were derived. To further evaluate the peptide delivery strategy, we analyzed the role of various kinases in IgE-mediated stimulation of mast cell exocytosis. Primary bone marrow-derived mast cells were transduced with retroviral constructs encoding short-kinase inhibitor motifs and analyzed by flow cytometry for effects on exocytosis. We found that a specific protein kinase A (PKA) inhibitor peptide suppressed IgE-mediated stimulation of mast cell exocytosis. This anti-exocytotic effect was mimicked by a small molecule inhibitor of PKA (KT5720). Thus, the ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention. We believe that such approaches might allow for new forms of gene therapy to become available.

Targeting rare populations of murine antigen-specific T lymphocytes by retroviral transduction for potential application in gene therapy for autoimmune disease.

CD4+ T cells are important mediators in the pathogenesis of autoimmunity and would therefore provide ideal candidates for lymphocyte-based gene therapy. However, the number of Ag-specific T cells in any single lesion of autoimmunity may be quite low. Successful gene transfer into autoantigen-specific CD4+ T cells would serve as an ideal vehicle for site-targeted gene therapy if it were possible to transduce preferentially the small number of autoantigen-specific T cells. In this study we have demonstrated that retroviral infection of CD4+ lymphocytes from either autoantigen-stimulated TCR transgenic mice, or Ag-activated immunized nontransgenic mice, with a retroviral vector (pGCIRES), resulted in the transduction of only the limited number of Ag-reactive CD4+ T cells. In contrast, polyclonal activation of the same cultures resulted in transduction of non-antigen-specific lymphocytes. Transduction of Ag-reactive CD4+ T cells with pGCIRES retrovirus encoding the regulatory genes IL-4 (IL4) and soluble TNF receptor (STNFR) resulted in stable integration and long-term expression of recombinant gene products. Moreover, expression of the pGCIRES marker protein, GFP, directly correlated with the expression of the upstream regulatory gene. Retroviral transduction of CD4+ T cells targeted specifically Ag-reactive cells and was cell cycle-dependent and evident only during the mitosis phase. These studies suggest that retroviral transduction of autoantigen-specific murine CD4+ T cells, using the pGCIRES retroviral vector, may provide a potential method to target and isolate the low frequency of autoantigen-specific murine CD4+ T cells, and provides a rational approach to gene therapy in animal models of autoimmunity.

Neurotrophin dependence domain: a domain required for the mediation of apoptosis by the p75 neurotrophin receptor.

The mechanisms underlying neurotrophin dependence, and cellular dependent states in general, are unknown. We show that a 29 amino acid region in the intracellular domain of the common neurotrophin receptor, p75NTR, is required for the mediation of apoptosis by p75NTR. Furthermore, contrary to results obtained with Fas, monomeric p75NTR is required for apoptosis induction, whereas multimerization inhibits the pro-apoptotic effect. Within the 29-residue domain required for apoptosis induction by p75NTR, a 14-residue region is sufficient as a peptide inducer of apoptosis. This 14-residue peptide requires the positively charged carboxyterminal residues for its effect on cell death, and these same residues are required by the full-length p75NTR. These studies define a novel type of domain that mediates neurotrophin dependence, and suggest that other cellular dependent states may be mediated by proteins displaying similar domains.