Virtual screening targeting the urokinase receptor (uPAR) led to (3R)-4-cyclohexyl-3-(hexahydrobenzo[d][1,3]dioxol-5-yl)-N-((hexahydrobenzo[d][1,3]dioxol-5-yl)methyl)butan-1-aminium 1 (IPR-1) and 4-(4-((3,5-dimethylcyclohexyl)carbamoyl)-2-(4-isopropylcyclohexyl)pyrazolidin-3-yl)piperidin-1-ium 3 (IPR-69). Synthesis of an analog of 1, namely 2 (IPR-9), and 3 led to breast MDA-MB-231 invasion, migration and adhesion assays with IC50 near 30 μM. Both compounds blocked angiogenesis with IC50 of 3 μM. Compounds 2 and 3 inhibited cell growth with IC50 of 6 and 18 μM and induced apoptosis. Biochemical assays revealed lead-like properties for 3, but not 2. Compound 3 administered orally reached peak concentration of nearly 40 μM with a half-life of about 2 hours. In NOD-SCID mice inoculated with breast TMD-231 cells in their mammary fat pads, compound 3 showed a 20% reduction in tumor volumes and less extensive metastasis was observed for the treated mice. The suitable pharmacokinetic properties of 3 and the encouraging preliminary results in metastasis make it an ideal starting point for next generation compounds.
Pancreatic cancer is an especially deadly form of cancer with a survival rate <2%. Pancreatic cancers respond poorly to existing chemotherapeutic agents and radiation, and progress for the treatment of pancreatic cancer remains elusive. To address this unmet medical need, a better understanding of critical pathways and molecular mechanisms involved in pancreatic tumor development, progression and resistance to traditional therapy is therefore critical. Reduction-oxidation (redox) signaling systems are emerging as important targets in pancreatic cancer. AP endonuclease1/ Redox effector factor 1 (APE1/Ref-1) is upregulated in human pancreatic cancer cells and modulation of its redox activity blocks the proliferation and migration of pancreatic cancer cells as well as pancreatic cancer-associated endothelial cells (PCECs) in vitro. Modulation of APE1/Ref-1 using a specific inhibitor of APE1/Ref-1’s redox function, E3330 leads to a decrease in transcription factor activity for NFκB, AP-1, and HIF1 in vitro. This study aims to further establish the redox signaling protein APE1/Ref-1 as a molecular target in pancreatic cancer. Here, we show that inhibition of APE1/Ref-1 via E3330 results in tumor growth inhibition in cell lines as well as pancreatic cancer xenograft models in mice. Pharmacokinetic (PK) studies also demonstrate that E3330 attains >10 μM blood concentrations and is detectable in tumor xenografts. Through inhibition of APE1/Ref-1, the activity of NFκB, AP-1, and HIF1α which are key transcriptional regulators involved in survival, invasion and metastasis is blocked. These data indicate that E3330, inhibitor of APE1/Ref-1, has potential in pancreatic cancer and clinical investigation of APE1/Ref-1 molecular target is warranted.
Pancreatic cancer; animal models of cancer; new targets; xenograft models; cellular responses to anticancer drugs; cellular pharmacology; pharmacokinetics and pharmacodynamics; agents with other mechanisms of action
Pre-clinical in vivo studies can help guide the selection of agents and regimens for clinical testing. However, one of the challenges in screening anti-cancer therapies is the assessment of off-target human toxicity. There is a need for in vivo models that can simulate efficacy and toxicities of promising therapeutic regimens. For example, hematopoietic cells of human origin are particularly sensitive to a variety of chemotherapeutic regimens but in vivo models to assess potential toxicities have not been developed. In this study, a xenograft model containing humanized bone marrow is utilized as an in vivo assay to monitor hematotoxicity.
A proof-of-concept, temozolomide-based regimen was developed that inhibits tumor xenograft growth. This regimen was selected for testing since it has been previously shown to cause myelosuppression in mice and humans. The dose-intensive regimen was administered to NOD/SCID/γchainnull mice reconstituted with human hematopoietic cells and the impact of treatment on human hematopoiesis was evaluated.
The dose-intensive regimen resulted in significant decreases in growth of human-glioblastoma xenografts. When this regimen was administered to mice containing humanized bone marrow, flow cytometric analyses indicated that the human bone-marrow cells were significantly more sensitive to treatment than the murine bone-marrow cells, and that the regimen was highly toxic to human-derived hematopoietic cells of all lineages (progenitor, lymphoid, and myeloid).
The humanized bone-marrow xenograft model described has the potential to be used as a platform for monitoring the impact of anti-cancer therapies on human hematopoiesis and could lead to subsequent refinement of therapies prior to clinical evaluation.
human xenograft model; myelosuppression; stem-cell
Cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) is a major resistance factor and critical anti-apoptotic regulator that inhibits tumor necrosis factor-alpha (TNF-alpha), Fas-L, and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis as well as chemotherapy-triggered apoptosis in malignant cells. c-FLIP is expressed as long (c-FLIPL), short (c-FLIPS), and c-FLIPR splice variants in human cells. c-FLIP binds to FADD and/or caspase-8 or -10 in a ligand-dependent and-independent fashion, which in turn prevents death-inducing signaling complex (DISC) formation and subsequent activation of the caspase cascade. Moreover, c-FLIPL and c-FLIPS are known to have multifunctional roles in various signaling pathways, as well as activating and/or upregulating several cytoprotective signaling molecules. Upregulation of c-FLIP has been found in various tumor types, and its downregulation has been shown to restore apoptosis triggered by cytokines and various chemotherapeutic agents. Hence, c-FLIP is an important target for cancer therapy. For example, small interfering RNAs (siRNAs) that specifically knockdown the expression of c-FLIPL in diverse human cancer cell lines augmented TRAIL-induced DISC recruitment and increased the efficacy of chemotherapeutic agents, thereby enhancing effector caspase stimulation and apoptosis. Moreover, small molecules causing degradation of c-FLIP as well as decreasing mRNA and protein levels of c-FLIPL and c-FLIPS splice variants have been found, and efforts are underway to develop other c-FLIP-targeted cancer therapies. This review focuses on (1) the functional role of c-FLIP splice variants in preventing apoptosis and inducing cytokine and drug resistance; (2) the molecular mechanisms that regulate c-FLIP expression; and (3) strategies to inhibit c-FLIP expression and function.
c-FLIP; apoptosis; death receptors; cancer; chemotherapy
Defining whether human circulating pro-angiogenic cells represent a subset of the hematopoietic system and express CD45 or are hematopoietic derivatives that do not express CD45 (and are called endothelial progenitor cells) remains controversial. We have previously developed a polychromatic flow cytometry (PFC) protocol to isolate subsets of hematopoietic cells and we now identify the circulating pool of CD34+CD45dim cells representing functional circulating hematopoietic stem and progenitor cells (CHSPCs) that can be separated on the basis of AC133 expression and report that the AC133+ subset of the CHSPCs enhances the growth of tumor blood vessels in vivo in immunodeficient mice. In addition, the ratio of AC133+ pro-angiogenic CHSPCs to AC133− non-angiogenic CHSPCs unambiguously correlates with the severity of the clinical state of patients with peripheral arterial disease. In sum, a PFC protocol validated via in vitro and in vivo analyses, can be used to interrogate the roles of human hematopoietic elements in the growth and maintenance of the vasculature.
Polychromatic flow cytometry; circulating progenitor cells; endothelial progenitor cells; angiogenesis; peripheral arterial disease
Humanized bone-marrow xenograft models that can monitor the long-term impact of gene-therapy strategies will help facilitate evaluation of clinical utility. The ability of the murine bone-marrow microenvironment in NOD/SCID versus NOD/SCID/γ chainnull mice to support long-term engraftment of MGMTP140K-transduced human-hematopoietic cells following alkylator-mediated in vivo selection was investigated. Mice were transplanted with MGMTP140K-transduced CD34+ cells and transduced cells selected in vivo. At 4 months after transplantation, levels of human-cell engraftment, and MGMTP140K-transduced cells in the bone marrow of NOD/SCID versus NSG mice varied slightly in vehicle- and drug-treated mice. In secondary transplants, although equal numbers of MGMTP140K-transduced human cells were transplanted, engraftment was significantly higher in NOD/SCID/γ chainnull mice compared to NOD/SCID mice at 2 months after transplantation. These data indicate that reconstitution of NOD/SCID/γ chainnull mice with human-hematopoietic cells represents a more promising model in which to test for genotoxicity and efficacy of strategies that focus on manipulation of long-term repopulating cells of human origin.
We recently reported that murine marrow cultured ex vivo for gamma-retrovirus transduction engrafts ~10 fold less well than fresh marrow upon transplantation into submyeloablated hosts. Here, we evaluated homing efficiency as a potential mechanism for this engraftment disparity, and whether CD26 inhibition with the tripeptide Diprotin A (DipA) would enhance engraftment of ex vivo cultured cells in submyeloablated hosts.
Homing and engraftment of fresh and ex vivo cultured lineage-negative (lin-) marrow cells in submyeloablated congenic hosts with and without DipA treatment was evaluated. Expression of CXCR4 and CD26 on fresh and cultured lin- marrow cells was compared.
Homing of lin- cells cultured for gamma-retrovirus transduction was ≥3-fold less than that of fresh lin- cells 20 hours after transplantation into submyeloablated hosts. DipA treatment of fresh lin- cells resulted in ≥-fold increased homing and engraftment in submyeloablated hosts. DipA treatment, however, did not significantly improve homing or engraftment of cells undergoing a three-day culture protocol for gamma-retrovirus transduction in submyeloablated hosts. CXCR4 expression on lin- cells was significantly decreased following three days of culture; CXCR4 expression was not significantly altered following overnight culture.
Ex vivo culture of lin- cells for gamma-retroviral transduction downregulates CXCR4 expression and markedly impairs homing and engraftment of murine lin- marrow in submyeloablated hosts. While inhibition of CD26 activity with DipA increases homing and engraftment of fresh lin- cells, DipA treatment does not improve homing and engraftment of cultured lin- marrow cells in submyeloablated congenic hosts.
The E3 ubiquitin ligase human murine double minute (HDM2) is overexpressed in 40%–80% of late-stage metastatic cancers in the absence of gene amplification. Hdm2 regulates p53 stability via ubiquitination and has also been implicated in altering the sensitivity of cells to TGF-β1. Whether TGF-β1 signaling induces Hdm2 expression leading to HDM2-mediated destabilization of p53 has not been investigated. In this study, we report that TGF-β1–activated SMA- and MAD3 (Smad3/4) transcription factors specifically bound to the second promoter region of HDM2, leading to increased HDM2 protein expression and destabilization of p53 in human cancer cell lines. Additionally, TGF-β1 expression led to Smad3 activation and murine double minute 2 (Mdm2) expression in murine mammary epithelial cells during epithelial-to-mesenchymal transition (EMT). Furthermore, histological analyses of human breast cancer samples demonstrated that approximately 65% of late-stage carcinomas were positive for activated Smad3 and HDM2, indicating a strong correlation between TGF-β1–mediated induction of HDM2 and late-stage tumor progression. Identification of Hdm2 as a downstream target of TGF-β1 represents a critical prosurvival mechanism in cancer progression and provides another point for therapeutic intervention in late-stage cancer.
Ultraviolet B (UVB) radiation causes cutaneous inflammation. One important clinical consequence of UVB-induced inflammation is increased pain or hyperalgesia, which is likely mediated by enhanced sensitivity of cutaneous sensory neurons. Previous studies have demonstrated that UVB radiation generates the lipid mediator, platelet-activating factor (PAF), as well as oxidized phospholipids that act as PAF-mimetics. These substances exert effects through the PAF receptor (PAF-R). This study was designed to assess whether PAF-R is involved in UVB-induced hyperalgesia. Intradermal injection of carbamoyl PAF (CPAF; 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine) resulted in an enhanced response to mechanical stimuli in wild-type mice but not in PAF-R knockout (KO) mice. There was no significant change in paw withdrawal to noxious thermal stimuli in either genotype after intradermal injection of CPAF. Exposure of the hind paw to 1,500 J m−2 UVB radiation caused an increased sensitivity to both mechanical and thermal stimulation in wild-type mice but not in PAF-R KO mice. The thermal hyperalgesia caused by UVB irradiation was inhibited in mice that lacked PAF-R in bone marrow-derived cells. These data demonstrate that the PAF-R is important for UVB-induced hyperalgesia. Further investigation of the role of PAF-R signaling in UVB-induced hyperalgesia could provide better understanding of the pathological processes initiated by UVB-induced skin damage.
Through its ability to both induce immunosuppression and act as a carcinogen, UVB radiation plays a major role in cutaneous malignancies. Recent studies have indicated that UVB-mediated inhibition of delayed-type hypersensitivity reactions is mediated, in part, by the lipid mediator platelet-activating factor (PAF). The objective of this study was to further define the mechanism by which UVB inhibits contact hypersensitivity (CHS) reactions. UVB irradiation resulted in an inhibition of subsequent CHS to the chemical DNFB in wild-type, but not in PAF-R-deficient mice. UVB-mediated inhibition of CHS was also blocked by a cyclooxygenase-2 (COX-2) inhibitor or a neutralizing antibody directed against IL-10. UVB irradiation upregulated IL-10 mRNA levels in lymph nodes and spleen only to significant levels in PAF-R-expressing mice. Bone marrow transplantation studies demonstrated that UVB-mediated immunomodulatory effects were dependent on PAF-R-positive bone marrow. These studies suggest that UVB irradiation results in epidermal production of PAF agonists, which then act on PAF-R-positive bone marrow-derived cells to upregulate IL-10 through COX-2-generated prostaglandins.
Using a clinically relevant transduction strategy, we investigated to what extent hematopoietic stem cells in lineage-negative bone marrow (Linneg BM) could be genetically modified with a FV vector that expresses the DNA repair protein, O6-methylguanine DNA methyltransferase (MGMTP140K) and selected in vivo with submyeloablative versus myeloablative alkylator therapy.
Linneg BM was transduced at a low multiplicity-of-infection (MOI), with the FV vector, MD9-P140K, that co-expresses MGMTP140K and the enhanced green fluorescent protein, transplanted into C57BL/6 mice, and mice treated with submyeloablative or myeloablative alkylator therapy. The BM was analyzed for the presence of in vivo selected, MD9-P140K-transduced cells at 6 months post-transplantation and subsequently transplanted into secondary recipient animals.
Following submyeloablative therapy, 55% of the mice expressed MGMTP140K in the BM. Proviral integration was observed in ∼50% of committed BM-derived progenitors and analysis of proviral insertion sites indicated up to 2 integrations per transduced progenitor colony. Transduced BM cells selected with submyeloablative therapy reconstituted secondary recipient mice for up to 6 months post-transplantation. In contrast, following delivery of myeloablative therapy to primary recipient mice, only 25% survived. Hematopoietic stem cells were transduced since BM cells from the surviving animals reconstituted secondary recipients with MGMTP140K positive cells for 5-6 months.
In vivo selection of MD9-P140K-transduced BM cells was more efficient following submyeloablative versus myeloablative therapy. These data indicate that a critical number of transduced-stem cells must be present to produce sufficient numbers of genetically modified progeny to protect against the acute toxicity associated with myeloablative therapy.
gene therapy; hematopoietic stem cells; foamy virus vector; O6-methylguanine DNA methyltransferase (MGMT)
Background. The use of 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) may help to establish the antitumor activity of enzastaurin, a novel protein kinase C-beta II (PKC-βII) inhibitor, in mouse xenografts. Methods. The hematologic cell line RAJI and the solid tumor cell line U87MG were each implanted in NOD/SCID mice. Standard tumor growth measurements and [18F]FDG PET imaging were performed weekly for up to three weeks after tumor implantation and growth. Results. Concomitant with caliper measurements, [18F]FDG PET imaging was performed to monitor glucose metabolism. Heterogeneity of glucose uptake in various areas of the tumors was observed after vehicle or enzastaurin treatment. This heterogeneity may limit the use of [18F]FDG PET imaging to measure enzastaurin-associated changes in xenograft tumors. Conclusion. [18F]FDG PET imaging technique does not correlate with standard caliper assessments in xenografts to assess the antitumor activity of enzastaurin. Future studies are needed to determine the use of [18F]FDG PET imaging in preclinical models.
The XPC protein (encoded by the xeroderma pigmentosum Xpc gene) is a key DNA damage recognition factor that is required for global genomic nucleotide excision repair (G-NER). In contrast to transcription-coupled nucleotide excision repair (TC-NER), XPC and G-NER have been reported to contribute only modestly to cell survival after DNA damage. Previous studies were conducted using fibroblasts of human or mouse origin. Since the advent of Xpc−/− mice, no study has focused on the bone marrow of these mice. We used carboplatin to induce DNA damage in Xpc−/− and strain-matched wild-type mice. Using several independent methods, Xpc−/− bone marrow was ∼10-fold more sensitive to carboplatin than the wild type. Importantly, 12/20 Xpc−/− mice died while 0/20 wild-type mice died. We conclude that G-NER, and XPC specifically, can contribute substantially to cell survival. The data are important in the context of cancer chemotherapy, where Xpc gene status and G-NER may be determinants of response to DNA-damaging agents including carboplatin. Additionally, altered cell cycles and altered DNA damage signalling may contribute to the cell survival end point.
Standard competitive repopulation assays have proven valuable in evaluating engraftment potential in ablated hosts, permitting comparisons between various test cell populations. However, no similar method exists to compare engraftment of test cells in submyeloablated hosts, which would be helpful given the applications of reduced-intensity conditioning for hematopoietic gene-replacement therapy and other cellular therapies. Here, we developed a novel assay to quantitate engraftment of hematopoietic stem cells in submyeloablated hosts.
Engraftment of murine marrow cells transduced with retroviral vectors using two separate protocols was compared to engraftment of fresh untreated competitor cells within low-dose radiation conditioned hosts using a “three-way” marking system, so that test, competitor and host cell chimerism could be reliably determined post-transplant.
We demonstrate that the repopulating ability of marrow cells transduced using two distinct protocols was reduced ~10-fold compared to fresh competitor cells in submyeloablated hosts utilizing the novel “three-way” transplant assay.
Murine marrow cells transduced using a clinically-applicable protocol acquire an engraftment defect in submyeloablated hosts, similar to cells transduced using a research protocol. We conclude that the submyeloablative competitive repopulation assay described here will be of benefit to comparatively assess the engraftment ability of manipulated hematopoietic stem cells using various culture protocols, such as to test the impact of modifications in transduction protocols needed to attain therapeutic levels of gene-corrected blood cells, or the effect of ex vivo expansion protocols on engraftment potential.
Engraftment; conditioning; submyeloablative; transduction; hematopoietic stem cells
Several factors are thought to limit the efficiency of retroviral transduction in clinical gene therapy protocols that target hematopoietic stem cells. For example, the level of expression of the amphotropic receptor Pit-2, a phosphate symporter, appears to be low in human and murine hematopoietic stem cells. We have previously demonstrated that transduction of hematopoietic cells in the presence of the fibronectin (FN) fragment CH-296 is extremely efficient (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876–882, 1996). To examine functionally whether the retrovirus receptor is a limiting factor in transduction of hematopoietic cells, we performed competition experiments in the presence of FN CH-296 with retrovirus vectors pseudotyped with the same or a different envelope protein. We demonstrate in both human erythroleukemia (HEL) cells and primary human CD34+ hematopoietic cells inhibition of efficient infection due to receptor interference when two vectors targeting the amphotropic receptor are used simultaneously. Receptor interference lasted up to 24 h. No interference was demonstrated when vectors targeting the amphotropic receptor and the gibbon ape leukemia virus (GALV) receptor Pit-1 were used concurrently. In contrast, simultaneous infection with vectors targeting both Pit-1 and Pit-2 yielded transduction efficiencies consistently higher than with either vector alone in both HEL cells and human CD34+ hematopoietic cells. These data demonstrate that the use of FN CH-296 leads to amphotropic receptor saturation in these cells. Simultaneous infection with vectors targeting both amphotropic and GALV receptors may prove to be of additional benefit in the design of gene therapy protocols.
Primary human T lymphocytes are powerful targets for genetic modification, although the use of these targets in human gene therapy protocols has been hampered by low levels of transduction. We have shown previously that significant increases in the transduction of hematopoietic stem and progenitor cells with retroviral vectors can be obtained by the colocalization of the retrovirus and target cells on specific fibronectin (FN) adhesion domains (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876–882, 1996). We studied the transfer of genes into primary T lymphocytes by using FN-assisted retroviral gene transfer. Activated T lymphocytes were infected for three consecutive days on the recombinant FN fragment CH-296 with a retroviral vector encoding the murine B7-1 protein. Transduced lymphocytes were analyzed for murine B7-1 expression, and it was found that under optimal conditions, 80 to 89% of the CD3+ lymphocytes were transduced. Gene transfer was predominantly augmented by the interaction between VLA-4 on the T lymphocytes and the FN adhesion site CS-1. Adenosine deaminase (ADA)-deficient primary T lymphocytes transduced on CH-296 with a retrovirus encoding murine ADA (mADA) exhibited levels of mADA activity severalfold higher than the levels of the endogenous human ADA protein observed in normal human T lymphocytes. Strikingly, the long-term expression of the transgene was dependent on the activation status of the lymphocytes. This approach will have important applications in human gene therapy protocols targeting primary T lymphocytes.