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Supplemental Figure 1. Cell surface expression of IL-11R in AML patient derived samples and hematopoietic cells from healthy bone marrow as analyzed by flow cytometry.

Supplemental Table 1. Features of synthesized analogs of BMTP-11 for drug-lead optimization.

Purpose: The IL11 receptor (IL11R) is an established molecular target in primary tumors of bone, such as osteosarcoma, and in secondary bone metastases from solid tumors, such as prostate cancer. However, its potential role in management of hematopoietic malignancies has not yet been determined. Here, we evaluated the IL11R as a candidate therapeutic target in human leukemia and lymphoma.Experimental Design and Results: First, we show that the IL11R protein is expressed in a variety of human leukemia– and lymphoma–derived cell lines and in a large panel of bone marrow samples from leukemia and lymphoma patients, whereas expression is absent from nonmalignant control bone marrow. Moreover, a targeted peptidomimetic prototype (termed BMTP-11), specifically bound to leukemia and lymphoma cell membranes, induced ligand–receptor internalization mediated by the IL11R, and resulted in a specific dose-dependent cell death induction in these cells. Finally, a pilot drug lead-optimization program yielded a new myristoylated BMTP-11 analogue with an apparent improved antileukemia cell profile.Conclusions: These results indicate (i) that the IL11R is a suitable cell surface target for ligand-directed applications in human leukemia and lymphoma and (ii) that BMTP-11 and its derivatives have translational potential against this group of malignant diseases. Clin Cancer Res; 21(13); 3041–51. ©2015 AACR.

Supplemental Figure 3. Drug activity of BMTP-11 and BMTP#4 on a panel of established leukemia and lymphoma cell lines.

Supplemental Figure Legends

Supplemental Figure 2. Cell membrane and cytoplasmic expression of IL-11R.

For two decades, the antigen recognized by the Pathologische Anatomie Leiden-Endothelium (PAL-E) monoclonal antibody, a standard vascular endothelial cell marker, has remained elusive. Here, we used a combinatorial phage display–based approach (“epitope mapping”) to select peptides binding to the original PAL-E antibody. We found that a subset of the selected panel of peptides had motifs with strong homology to an exposed site within the b1 domain of human neuropilin-1 (NRP-1). We confirmed peptide binding by ELISA and by surface plasmon resonance. We also showed that the PAL-E antigen colocalizes with NRP-1 staining in endothelial cells. Crystal structure of the b1 domain in NRP-1 suggests that the PAL-E binding site overlaps with a vascular endothelial growth factor (VEGF) binding site. Taken together, these results indicate that NRP-1 is an endothelial cell antigen recognized by the true PAL-E antibody. The consistent biochemical, morphologic, and functional features between the PAL-E antigen and NRP-1 support our interpretation. Given that NRP-1 is a VEGF receptor, these results explain the attributes of the PAL-E antibody as a marker of vascular permeability and angiogenesis. [Cancer Res 2007;67(20):9623–9]

Supplementary Figure S1 from Ligand-Directed Surface Profiling of Human Cancer Cells with Combinatorial Peptide Libraries

A collection of 60 cell lines derived from human tumors (NCI-60) has been widely explored as a tool for anticancer drug discovery. Here, we profiled the cell surface of the NCI-60 by high-throughput screening of a phage-displayed random peptide library and classified the cell lines according to the binding selectivity of 26,031 recovered tripeptide motifs. By analyzing selected cell-homing peptide motifs and their NCI-60 recognition patterns, we established that some of these motifs (a) are similar to domains of human proteins known as ligands for tumor cell receptors and (b) segregate among the NCI-60 in a pattern correlating with expression profiles of the corresponding receptors. We biochemically validated some of the motifs as mimic peptides of native ligands for the epidermal growth factor receptor. Our results indicate that ligand-directed profiling of tumor cell lines can select functional peptides from combinatorial libraries based on the expression of tumor cell surface molecules, which in turn could be exploited as “druggable” receptors in specific types of cancer. (Cancer Res 2006; 66(1): 34-40)

Supplementary Table S1 from Ligand-Directed Surface Profiling of Human Cancer Cells with Combinatorial Peptide Libraries

Supplementary Tables 1-2 from The Original Pathologische Anatomie Leiden-Endothelium Monoclonal Antibody Recognizes a Vascular Endothelial Growth Factor–Binding Site within Neuropilin-1

Supplementary Legend and Methods from Ligand-Directed Surface Profiling of Human Cancer Cells with Combinatorial Peptide Libraries

Lower invertebrates' hearts such as those of zebrafish have the capacity for scarless myocardial regeneration which is lost by mammalian hearts as they form a fibrotic scar tissue instead of regenerating the injured area. However, neonatal mammalian hearts have a remarkable capacity for regeneration highlighting conserved evolutionary mechanisms underlying such a process. Studies investigated the underlying mechanism of myocardial regeneration in species capable to do so, to see its applicability on mammals. The epicardium, the mesothelial outer layer of the vertebrate heart, has proven to play an important role in the process of repair and regeneration. It serves as an important source of smooth muscle cells, cardiac fibroblasts, endothelial cells, stem cells, and signaling molecules that are involved in this process. Here we review the role of the epicardium in myocardial regeneration focusing on the different involved; Activation, epithelial to mesenchymal transition, and differentiation. In addition, we will discuss its contributory role to different aspects that support myocardial regeneration. Of these we will discuss angiogenesis and the formation of a regenerate extracellular matrix. Moreover, we will discuss several factors that act on the epicardium to affect regeneration. Finally, we will highlight the utility of the epicardium as a mode of cell therapy in the treatment of myocardial injury.

Mutations in the LMNA gene, which encodes for the nuclear lamina proteins lamins A and C, are responsible for a diverse group of diseases known as laminopathies. One type of laminopathy is Dilated Cardiomyopathy (DCM), a heart muscle disease characterized by dilation of the left ventricle and impaired systolic function, often leading to heart failure and sudden cardiac death. LMNA is the second most commonly mutated gene in DCM. In addition to LMNA, mutations in more than 60 genes have been associated with DCM. The DCM-associated genes encode a variety of proteins including transcription factors, cytoskeletal, Ca2+-regulating, ion-channel, desmosomal, sarcomeric, and nuclear-membrane proteins. Another important category among DCM-causing genes emerged upon the identification of DCM-causing mutations in RNA binding motif protein 20 (RBM20), an alternative splicing factor that is chiefly expressed in the heart. In addition to RBM20, several essential splicing factors were validated, by employing mouse knock out models, to be embryonically lethal due to aberrant cardiogenesis. Furthermore, heart-specific deletion of some of these splicing factors was found to result in aberrant splicing of their targets and DCM development. In addition to splicing alterations, advances in next generation sequencing highlighted the association between splice-site mutations in several genes and DCM. This review summarizes LMNA mutations and splicing alterations in DCM and discusses how the interaction between LMNA and splicing regulators could possibly explain DCM disease mechanisms.

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D (KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.

Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome. Most LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and altered interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes. Here we report in mice that lamin-A/C-deficient (Lmna(-/-)) and Lmna(N195K/N195K) mutant cells have impaired nuclear translocation and downstream signalling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function. Altered nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna(-/-) and Lmna(N195K/N195K) mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease aetiology for the cardiac phenotype in many laminopathies, whereby lamin A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization.

Lamins are intermediate filament proteins that assemble into a meshwork underneath the inner nuclear membrane, the nuclear lamina. Mutations in the LMNA gene, encoding lamins A and C, cause a variety of diseases collectively called laminopathies. The disease mechanism for these diverse conditions is not well understood. Since lamins A and C are fundamental determinants of nuclear structure and stability, we tested whether defects in nuclear mechanics could contribute to the disease development, especially in laminopathies affecting mechanically stressed tissue such as muscle. Using skin fibroblasts from laminopathy patients and lamin A/C-deficient mouse embryonic fibroblasts stably expressing a broad panel of laminopathic lamin A mutations, we found that several mutations associated with muscular dystrophy and dilated cardiomyopathy resulted in more deformable nuclei; in contrast, lamin mutants responsible for diseases without muscular phenotypes did not alter nuclear deformability. We confirmed our results in intact muscle tissue, demonstrating that nuclei of transgenic Drosophila melanogaster muscle expressing myopathic lamin mutations deformed more under applied strain than controls. In vivo and in vitro studies indicated that the loss of nuclear stiffness resulted from impaired assembly of mutant lamins into the nuclear lamina. Although only a subset of lamin mutations associated with muscular diseases caused increased nuclear deformability, almost all mutations tested had defects in force transmission between the nucleus and cytoskeleton. In conclusion, our results indicate that although defective nuclear stability may play a role in the development of muscle diseases, other factors, such as impaired nucleo-cytoskeletal coupling, likely contribute to the muscle phenotype.

Maintaining physical connections between the nucleus and the cytoskeleton is important for many cellular processes that require coordinated movement and positioning of the nucleus. Nucleo-cytoskeletal coupling is also necessary to transmit extracellular mechanical stimuli across the cytoskeleton to the nucleus, where they may initiate mechanotransduction events. The LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, formed by the interaction of nesprins and SUN proteins at the nuclear envelope, can bind to nuclear and cytoskeletal elements; however, its functional importance in transmitting intracellular forces has never been directly tested. This question is particularly relevant since recent findings have linked nesprin mutations to muscular dystrophy and dilated cardiomyopathy. Using biophysical assays to assess intracellular force transmission and associated cellular functions, we identified the LINC complex as a critical component for nucleo-cytoskeletal force transmission. Disruption of the LINC complex caused impaired propagation of intracellular forces and disturbed organization of the perinuclear actin and intermediate filament networks. Although mechanically induced activation of mechanosensitive genes was normal (suggesting that nuclear deformation is not required for mechanotransduction signaling) cells exhibited other severe functional defects after LINC complex disruption; nuclear positioning and cell polarization were impaired in migrating cells and in cells plated on micropatterned substrates, and cell migration speed and persistence time were significantly reduced. Taken together, our findings suggest that the LINC complex is critical for nucleo-cytoskeletal force transmission and that LINC complex disruption can result in defects in cellular structure and function that may contribute to the development of muscular dystrophies and cardiomyopathies.

Therapies selectively targeting ischemic myocardium could be applied by intravenous injection. Here, we report an approach for ischemic tissue-selective targeting based on in vivo screening of random peptide sequences using phage display. We performed in vivo biopanning using a phage library in a rat model of ischemia-reperfusion and identified three peptide motifs, CSTSMLKAC, CKPGTSSYC, and CPDRSVNNC, that exhibited preferential binding to ischemic heart tissue compared to normal heart as well as other control organs. The CSTSMLKAC sequence was capable of mediating selective homing of phage to ischemic heart tissue. The CSTSMLKAC peptide was then made as a fusion protein with Sumo-mCherry and injected intravenously in a mouse model of myocardial ischemia-reperfusion injury; subsequently, bio-distribution of Sumo-mCherry-CSTSMLKAC was measured with quantitative ELISA. The targeting peptide led to a significant increase in homing to ischemic left ventricle compared to tissues from non-ischemic left ventricle, the right ventricle, lung, liver, spleen, skeletal muscle, and brain (all p

Targeted drug delivery offers an opportunity for the development of safer and more effective therapies for the treatment of cancer. In this study, we sought to identify short, cell-internalizing peptide ligands that could serve as directive agents for specific drug delivery in hematologic malignancies. By screening of human leukemia cells with a combinatorial phage display peptide library, we isolated a peptide motif, sequence Phe-Phe/Tyr-Any-Leu-Arg-Ser (FF/YXLRS), which bound to different leukemia cell lines and to patient-derived bone marrow samples. The motif was internalized through a receptor-mediated pathway, and we next identified the corresponding receptor as the transmembrane glycoprotein neuropilin-1 (NRP-1). Moreover, we observed a potent anti-leukemia cell effect when the targeting motif was synthesized in tandem to the pro-apoptotic sequence D(KLAKLAK)2. Finally, our results confirmed increased expression of NRP-1 in representative human leukemia and lymphoma cell lines and in a panel of bone marrow specimens obtained from patients with acute lymphoblastic leukemia or acute myelogenous leukemia compared with normal bone marrow. These results indicate that NRP-1 could potentially be used as a target for ligand-directed therapy in human leukemias and lymphomas and that the prototype CGFYWLRSC-GG-D(KLAKLAK)2 is a promising drug candidate in this setting.

Cardiomyopathies are among the leading causes of premature sudden death. Their etiology is genetically heterogeneous with more than 50 genes linked to them. The most substantial mutations involved in the cardiac phenotypes are those affecting the integrity and structure of the nuclear lamina; LMNA

Acute myelogenous leukemias (AMLs) are characterized by medullary and extramedullary invasion. We hypothesized that a supramolecular complex, the leukemia-cell invadosome, which contains certain integrins, matrix metalloproteinases (MMPs), and other as-yet unidentified proteins, is essential for tissue invasion and may be central to the phenotypic diversity observed in the clinic. Here we show that the specific binding of MMP-9 to leukocyte surface β2 integrin is required for pericellular proteolysis and migration of AML-derived cells. An efficient antileukemia effect was obtained by the hexapeptide HFDDDE, a motif of the MMP-9 catalytic domain that mediates integrin binding: HFDDDE prevented proMMP-9 binding, transmigration through a human endothelial cell layer, and extracellular matrix degradation. Notably, the functional protein anchorage between β2 integrin and proMMP-9 described in this study does not involve the enzymatic active sites targeted by known MMP inhibitors. Taken together, our results provide a biochemical working definition for the human leukemia invadosome. Disruption of specific protein complexes within this supramolecular target complex may yield a new class of anti-AML drugs with anti-invasion (rather than or in addition to cytotoxic) attributes.

Ligand-directed delivery of agents to leukemia and lymphoma cells has the potential to yield new mechanistic disease insights and targeted therapies. Here we set out to target the macropinocytotic pathway with a combinatorial approach. From the screening of acute T-lymphoblastic leukemia Molt-4 cells with a random phage-display peptide library, we isolated a phage displaying the sequence CAYHRLRRC. This peptide contains a lymph node-homing motif (Cys-Ala-Tyr) and a cell-penetrating motif (Arg-Leu-Arg-Arg). Binding of this ligand-directed phage to a large panel of leukemia/lymphoma cells and to patient-derived samples was much higher than to non-leukemia control cells. CAYHRLRRC phage internalization into Molt-4 cells is both energy- and temperature-dependent. Flow cytometry with fluorescein-labeled peptide and endocytosis blocking with specific inhibitors revealed that CAYHRLRRC is indeed taken up through macropinocytosis in Molt-4 and K562 human leukemia cells. Unexpectedly, the cell surface receptor for the CAYHRLRRC peptide is not a heparan sulfate proteoglycan as it would be predicted for other cell-penetrating peptides. Confirming this interpretation, a CAYHRLRRC-directed peptidomimetic-induced cell death in all the leukemia and lymphoma cells was evaluated, whereas a control transactivator of transcription protein (tat)-directed proapoptotic peptidomimetic was non-selective. In summary, the targeting peptide CAYHRLRRC is selectively internalized through macropinocytosis in leukemia and lymphoma cells and has potential as a drug lead for ligand-directed anti-leukemia therapies.

A replication-defective, vesicular stomatitis virus G-pseudotyped, Moloney murine leukemia virus retroviral vector (vLTR-IGF-IR(AS)) was generated in which a type I insulin-like growth factor receptor (IGF-IR) antisense fragment is expressed in a bicistronic mRNA with an enhanced green fluorescent protein (EGFP) reporter under the control of a potent long terminal repeat (LTR). The suitability of these retroparticles for gene therapy was tested with highly metastatic, carcinoma H-59 cells, which depend on IGF-IR expression for tumorigenicity and metastasis. Transduction with these, but not with control retroviral particles expressing EGFP only, resulted in a 70% reduction in IGF-IR levels and the loss of IGF-IR-regulated functions. Moreover, the ability of vLTR-IGF-IR(AS) retroparticle-transduced tumor cells to form experimental hepatic metastases was significantly reduced relative to controls. The results identify retrovector-mediated delivery of IGF-IR antisense as a potential strategy for cancer gene therapy.

Purpose: The IL11 receptor (IL11R) is an established molecular target in primary tumors of bone, such as osteosarcoma, and in secondary bone metastases from solid tumors, such as prostate cancer. However, its potential role in management of hematopoietic malignancies has not yet been determined. Here, we evaluated the IL11R as a candidate therapeutic target in human leukemia and lymphoma. Experimental Design and Results: First, we show that the IL11R protein is expressed in a variety of human leukemia– and lymphoma–derived cell lines and in a large panel of bone marrow samples from leukemia and lymphoma patients, whereas expression is absent from nonmalignant control bone marrow. Moreover, a targeted peptidomimetic prototype (termed BMTP-11), specifically bound to leukemia and lymphoma cell membranes, induced ligand–receptor internalization mediated by the IL11R, and resulted in a specific dose-dependent cell death induction in these cells. Finally, a pilot drug lead-optimization program yielded a new myristoylated BMTP-11 analogue with an apparent improved antileukemia cell profile. Conclusions: These results indicate (i) that the IL11R is a suitable cell surface target for ligand-directed applications in human leukemia and lymphoma and (ii) that BMTP-11 and its derivatives have translational potential against this group of malignant diseases. Clin Cancer Res; 21(13); 3041–51. ©2015 AACR.

Transplantable bone marrow stromal cells can be utilized for cell therapy of mesenchymal disorders. They can also be genetically engineered to express synthetic transgenes and subsequently serve as a platform for systemic delivery of therapeutic proteins in vivo. Inducible production of therapeutic proteins would markedly enhance the usefulness of stromal cells for cell therapy applications. We determined whether synthetic corticosteroid hormones can be used to tightly control transgene expression via the glucocorticoid response pathway in primary bone marrow stromal cells. This regulatory mechanism does not require the presence of potentially immunogenic prokaryotic or chimeric "Trans-activators." Further, synthetic corticosteroids are pharmaceutical agents that can be readily used in vivo. We designed a self-inactivating retroviral vector in which expression of the green fluorescent protein (GFP) reporter is controlled by a minimal synthetic promoter composed of five tandem glucocorticoid response elements upstream of a TATAA box. Vesicular stomatitis virus G-pseudotyped retroparticles were synthesized and utilized to transduce cultured cell lines and primary rat bone marrow stromal cells. We have shown that primary rat bone marrow stromal cells could be efficiently engineered with our GRE-containing retrovector, basal reporter expression was low in the absence of exogenous synthetic corticosteroids, and GFP expression was dexamethasone inducible and reversible. To summarize, this strategy allows dexamethasone-induced, "on-demand" transgene expression from transplantable genetically engineered bone marrow stromal cells.

Neutrophils are characterized by their distinct nuclear shape, which is thought to facilitate the transit of these cells through pore spaces less than one-fifth of their diameter. We used human promyelocytic leukemia (HL-60) cells as a model system to investigate the effect of nuclear shape in whole cell deformability. We probed neutrophil-differentiated HL-60 cells lacking expression of lamin B receptor, which fail to develop lobulated nuclei during granulopoiesis and present an in vitro model for Pelger-Huet anomaly; despite the circular morphology of their nuclei, the cells passed through micron-scale constrictions on similar timescales as scrambled controls. We then investigated the unique nuclear envelope composition of neutrophil-differentiated HL-60 cells, which may also impact their deformability; although lamin A is typically down-regulated during granulopoiesis, we genetically modified HL-60 cells to generate a subpopulation of cells with well defined levels of ectopic lamin A. The lamin A-overexpressing neutrophil-type cells showed similar functional characteristics as the mock controls, but they had an impaired ability to pass through micron-scale constrictions. Our results suggest that levels of lamin A have a marked effect on the ability of neutrophils to passage through micron-scale constrictions, whereas the unusual multilobed shape of the neutrophil nucleus is less essential.

Cells sense their physical environment through mechanotransduction–that is, by translating mechanical forces and deformations into biochemical signals such as changes in intracellular calcium concentration or activation of diverse signalling pathways. In turn, these signals can adjust cellular and extracellular structure. This mechanosensitive feedback modulates cellular functions as diverse as migration, proliferation, differentiation, and apoptosis and is critical for organ development and homeostasis. Consequently, defects in mechanotransduction–often caused by mutations or misregulation of proteins that disturb cellular or extracellular mechanics–are implicated in the development of a wide array of diseases, ranging from muscular dystrophies and cardiomyopathies to cancer progression and metastasis.

For two decades, the antigen recognized by the Pathologische Anatomie Leiden-Endothelium (PAL-E) monoclonal antibody, a standard vascular endothelial cell marker, has remained elusive. Here, we used a combinatorial phage display–based approach (“epitope mapping”) to select peptides binding to the original PAL-E antibody. We found that a subset of the selected panel of peptides had motifs with strong homology to an exposed site within the b1 domain of human neuropilin-1 (NRP-1). We confirmed peptide binding by ELISA and by surface plasmon resonance. We also showed that the PAL-E antigen colocalizes with NRP-1 staining in endothelial cells. Crystal structure of the b1 domain in NRP-1 suggests that the PAL-E binding site overlaps with a vascular endothelial growth factor (VEGF) binding site. Taken together, these results indicate that NRP-1 is an endothelial cell antigen recognized by the true PAL-E antibody. The consistent biochemical, morphologic, and functional features between the PAL-E antigen and NRP-1 support our interpretation. Given that NRP-1 is a VEGF receptor, these results explain the attributes of the PAL-E antibody as a marker of vascular permeability and angiogenesis. [Cancer Res 2007;67(20):9623–9]

BackgroundT-cell activation leads to signaling pathways that ultimately result in induction of gene transcription from the interleukin-2 (IL-2) promoter. We hypothesized that the IL-2 promoter or its synthetic derivatives can lead to T-cell specific, activation-induced transgene expression. Our objective was to develop a retroviral vector for stable and activation-induced transgene expression in T-lymphocytes.ResultsFirst, we compared the transcriptional potency of the full-length IL-2 promoter with that of a synthetic promoter composed of 3 repeats of the Nuclear Factor of Activated T-Cells (NFAT) element following activation of transfected Jurkat T-cells expressing the large SV40 T antigen (Jurkat TAg). Although the NFAT3 promoter resulted in a stronger induction of luciferase reporter expression post stimulation, the basal levels of the IL-2 promoter-driven reporter expression were much lower indicating that the IL-2 promoter can serve as a more stringent activation-dependent promoter in T-cells. Based on this data, we generated a self-inactivating retroviral vector with the full-length human IL-2 promoter, namely SINIL-2pr that incorporated the enhanced green fluorescent protein (EGFP) fused to herpes simplex virus thymidine kinase as a reporter/suicide "bifunctional" gene. Subsequently, Vesicular Stomatitis Virus-G Protein pseudotyped retroparticles were generated for SINIL-2pr and used to transduce the Jurkat T-cell line and the ZAP-70-deficient P116 cell line. Flow cytometry analysis showed that EGFP expression was markedly enhanced post co-stimulation of the gene-modified cells with 1 μM ionomycin and 10 ng/ml phorbol 12-myristate 13-acetate (PMA). This activation-induced expression was abrogated when the cells were pretreated with 300 nM cyclosporin A.ConclusionThese results demonstrate that the SINIL-2pr retrovector leads to activation-inducible transgene expression in Jurkat T-cell lines. We propose that this design can be potentially exploited in several cellular immunotherapy applications.

Background Self-inactivating retroviral vectors (SIN) are often associated with very low titers. Promoter elements embedded within SIN designs may suppress transcription of packageable retroviral RNA which in turn results in titer reduction. We tested whether this dominant-negative effect involves histone acetylation state. We designed an MLV-derived SIN vector using the cytomegalovirus immediate early enhancer-promoter (CMVIE) as an embedded internal promoter (SINCMV) and transfected the pantropic 293GPG packaging cell line. Results The SINCMV retroviral producer had uniformly very low titers (~10,000 infectious retroparticles per ml). Northern blot showed low levels of expression of retroviral mRNA in producer cells in particular that of packageable RNA transcript. Treatment of the producers with the histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A reversed transcriptional suppression and resulted in an average 106.3 ± 4.6 – fold (P = 0.002) and 15.5 ± 1.3 – fold increase in titer (P = 0.008), respectively. A histone gel assay confirmed increased histone acetylation in treated producer cells. Conclusion These results show that SIN retrovectors incorporating strong internal promoters such as CMVIE, are susceptible to transcriptional silencing and that treatment of the producer cells with HDAC inhibitors can overcome this blockade suggesting that histone deacetylation is implicated in the mechanism of transcriptional suppression.

A collection of 60 cell lines derived from human tumors (NCI-60) has been widely explored as a tool for anticancer drug discovery. Here, we profiled the cell surface of the NCI-60 by high-throughput screening of a phage-displayed random peptide library and classified the cell lines according to the binding selectivity of 26,031 recovered tripeptide motifs. By analyzing selected cell-homing peptide motifs and their NCI-60 recognition patterns, we established that some of these motifs (a) are similar to domains of human proteins known as ligands for tumor cell receptors and (b) segregate among the NCI-60 in a pattern correlating with expression profiles of the corresponding receptors. We biochemically validated some of the motifs as mimic peptides of native ligands for the epidermal growth factor receptor. Our results indicate that ligand-directed profiling of tumor cell lines can select functional peptides from combinatorial libraries based on the expression of tumor cell surface molecules, which in turn could be exploited as “druggable” receptors in specific types of cancer. (Cancer Res 2006; 66(1): 34-40)

Vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped replication-defective retroviral particles are pantropic and amenable to concentration to high titer by ultracentrifugation. These features have allowed development of effective retroviral transduction protocols for stem cells in vitro as well as for tissue engineering in vivo. However, retroparticle ultracentrifugation protocols will also copellet cellular and subcellular debris released from retroviral producer cell lines during vector manufacture. We have analyzed concentrated vector preparations by chromatography and have found that a significant amount of genomic DNA released from producer cells coconcentrates with retroviral particles. In an effort to generate high-purity retroparticle preparations, devoid of subcellular contaminants and contaminating genomic DNA, we have developed a process using size-exclusion chromatography combined with host cell nucleic acid digestion and concentration by ultrafiltration. The procedure allowed for a final recovery of 19 +/- 0.4% infectious viral particles from unfractionated starting material, with an average retroparticle concentration of 7.7 x 10(7) +/- 1.5 x 10(6)/ml. The intact virus is of high purity, >90% as determined by anion-exchange high-performance liquid chromatography. Retroparticle structure appeared intact as determined by negative stain electron microscopy and purified virus was functional and allowed for efficient transduction of primary human bone marrow stromal cells in vitro. In conclusion, we have developed a VSV-G retrovector purification process that can be applied to large-scale retroviral production ideal for cell and gene therapy applications.

Expression vectors encoding herpes simplex virus thymidine kinase (HSVTK) have been extensively used in cell and gene therapy applications either as anticancer "suicide" or as "self-destruct" transgenes in adoptive immunotherapy applications. In both gene therapy applications, reliable detection of HSVTK transgene expression is required in genetically engineered cells. Direct fluorescent labeling of the HSVTK protein may be the remedy. We designed a retrovector encoding a chimeric GFP-HSVTK fusion protein that can serve as a bifunctional suicide and reporter transgene. The fusion gene was incorporated in a VSV G-pseudotyped retrovector (vGFPTKfus) and high-titer stable retroviral producer was generated ( approximately 3 x 10(6) retroparticles/ml). Tumor cell lines transduced at an MOI of 8 for 3 days led to >90% gene transfer efficiency. Southern blot analysis confirmed that unrearranged proviral genomes integrated in chromosomal DNA. Protein extract immunoblot with HSVTK antisera revealed the presence of a 70-kDa protein consistent with the predicted size of an HSVTK-GFP fusion protein. Fluorescence microscopy and FACS analysis revealed that GFPTKfus-mediated fluorescence was nuclear localized and was 30-fold greater than that observed in a bicistronic HSVTK-GFP vector. Growth of cell lines expressing vGFPTKfus was significantly suppressed in the presence of ganciclovir. The DA3 mouse mammary carcinoma cell line was transduced with vGFPTKfus and implanted in syngeneic BALB/c mice. Preestablished tumors completely regressed in seven of nine mice treated with ganciclovir. Normal human peripheral blood T lymphocytes were transduced with vGFPTKfus and nucleus-restricted green fluorescence was observed. Sorting of green fluorescent lymphocytes allowed for selection of engineered cells. In conclusion, we demonstrate the utility of vGFPTKfus as a suicide/reporter transgene in tumor cells in vitro and in vivo. Furthermore, its potential use as an analytical and therapeutic tool targeting human T lymphocytes is shown.

Targeted drug delivery offers an opportunity for the development of safer and more effective therapies for the treatment of cancer. In this study, we sought to identify short cell-internalizing peptide ligands that bind to specific cell surface receptors on malignant hematopoietic cells. Such targeting motifs could also serve as vehicles for preferential delivery of cytotoxic agents to leukemias and lymphomas. By screening of human leukemia cells with a combinatorial phage display peptide library, we isolated a peptide motif, sequence FF/YXLRS, which bound to human leukemia and lymphoma cell lines as well as to primary human acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) bone marrow specimens obtained from patients. The motif was internalized through a receptor-mediated pathway, and we next identified the corresponding receptor as the transmembrane glycoprotein neuropilin-1 (NRP-1). Moreover, the functional relevance of this internalizing receptor was evaluated in the context of disease progression via the targeted delivery of a pro-apoptotic peptidomimetic to leukemia and lymphoma cells, and a potent anti-leukemia cell effect was observed both with cell lines and patient samples when the targeting motif was synthesized in tandem to the pro-apoptotic sequence D(KLAKLAK)2. Markedly, the pro-apoptotic peptide did not have an effect on purified mononuclear cells from normal bone marrow cells. Finally, our results confirmed increased expression of NRP-1 in representative human leukemia and lymphoma cell lines and in a panel of bone marrow specimens obtained from patients with ALL or AML compared to normal bone marrow specimens. Taken together, our results indicate that NRP-1 could, potentially, be used as a target for ligand-directed therapy in human leukemias and lymphomas - as well as in many human solid tumors, which are also know to over-express NRP-1 - and that the prototype CGFYWLRSC-GG-D(KLAKLAK)2 is a promising drug candidate in this setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2903. doi:1538-7445.AM2012-2903

The ability of cells to deform through narrow spaces is critical for processes ranging from immune function to metastasis. Neutrophils are the most abundant white blood cell, which are required to transit through spaces less than 1/5 of the cell's diameter. As the nucleus is typically the stiffest organelle in the cell, the lobulated shape of the neutrophil nucleus is thought to facilitate its transit. However, neither the mechanical properties of the nuclei, nor the mechanism underlying the transition from ovoid to lobulated nuclear shape, are fully understood. We used HL60 cells as a differentiable model system to study nuclear shape transitions and the effects on cell mechanics. To elucidate the effects of the nuclear envelope protein, lamin A, we genetically modified the cells to generate subpopulations of cells with well-defined lamin A levels. Quantitative image analysis revealed that increased lamin A expression inhibits the nuclear shape transition to the typical lobulated morphology. To determine the effect on whole-cell mechanics, we measured cell deformability by flowing cells through channels of a microfluidic device, monitoring transit time and nuclear deformation. In addition, we performed functional assays to test if the impaired transition in nuclear morphology is also associated with defects in phagocytotic function, and thus reflect overall impairment of differentiation due to increased lamin levels. These results help to elucidate the molecular mechanism of granulocyte differentiation, and may have possible implications for understanding reduced immune function in aging, where lamin A has been reported to accumulate at the nuclear envelope.