Anthony L. Sinn

UVB Radiation-Mediated Inhibition of Contact Hypersensitivity Reactions Is Dependent on the Platelet-Activating Factor System

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.

Impact of APE1/Ref-1 Redox Inhibition on Pancreatic Tumor Growth

Pancreatic cancer is especially a deadly form of cancer with a survival rate less than 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 and pancreatic cancer-associated endothelial cells 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 HIF1α 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 and pancreatic cancer xenograft models in mice. Pharmacokinetic studies also show that E3330 attains more than10 μmol/L blood concentrations and is detectable in tumor xenografts. Through inhibition of APE1/Ref-1, the activity of NFκB, AP-1, and HIF1α that 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. Mol Cancer Ther; 10(9); 1698–708. ©2011 AACR.

Small-molecule inhibition of the uPAR·uPA interaction: synthesis, biochemical, cellular, in vivo pharmacokinetics and efficacy studies in breast cancer metastasis.

The uPAR·uPA protein-protein interaction (PPI) is involved in signaling and proteolytic events that promote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational screening of a commercial chemical library and shown that the compound inhibited uPAR·uPA PPI in competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show, using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to uPAR with sub-micromolar affinity of 0.2 μM. We show that 4 blocks invasion of breast MDA-MB-231, and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives of 4 also inhibited MMP activity and blocked invasion in a concentration-dependent manner. Compound 4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID mice revealed a half-life of nearly 5h and peak concentration of 5 μM. Similar levels of the inhibitor were detected in tumor tissue up to 10h. Female NSG mice inoculated with highly malignant TMD-MDA-MB-231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the treated mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a promising template for the development of compounds with enhanced PK parameters and greater efficacy.

Humanized Bone Marrow Mouse Model as a Preclinical Tool to Assess Therapy-Mediated Hematotoxicity

Purpose: Preclinical in vivo studies can help guide the selection of agents and regimens for clinical testing. However, one of the challenges in screening anticancer 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. Experimental Design: A proof-of-concept, temozolomide-based regimen was developed that inhibits tumor xenograft growth. This regimen was selected for testing because it has been previously shown to cause myelosuppression in mice and humans. The dose-intensive regimen was administered to NOD.Cg-PrkdcscidIL2rgtm1Wjl/Sz (NOD/SCID/γchainnull), reconstituted with human hematopoietic cells, and the impact of treatment on human hematopoiesis was evaluated. Results: 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). Conclusions: The humanized bone marrow xenograft model described has the potential to be used as a platform for monitoring the impact of anticancer therapies on human hematopoiesis and could lead to subsequent refinement of therapies prior to clinical evaluation. Clin Cancer Res; 17(8); 2195–206. ©2011 AACR.

Soypeptide lunasin in cytokine immunotherapy for lymphoma.

Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We have previously reported that post-transplant lymphoma patients have an acquired deficiency of signal transducer and activator of transcription 4, which results in defective IFNγ production during clinical immunotherapy. With the goal of further improving cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that synergistically works with cytokines on natural killer (NK) cells. Peripheral blood mononuclear cells of healthy donors and post-transplant lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine. Adding lunasin to IL-12- or IL-2-stimulated NK cells demonstrated synergistic effects in the induction of IFNG and GZMB involved in cytotoxicity. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNγ production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines displayed higher tumoricidal activity than those stimulated with cytokines alone using in vitro and in vivo tumor models. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation on target gene loci. Lunasin represents a different class of immune modulating agent that may augment the therapeutic responses mediated by cytokine-based immunotherapy.

In vivo selection of hematopoietic stem cells transduced at a low multiplicity-of-infection with a foamy viral MGMTP140K vector

OBJECTIVE Using a clinically relevant transduction strategy, we investigated to what extent hematopoietic stem cells in lineage-negative bone marrow (Lin(neg) BM) could be genetically modified with an foamy virus (FV) vector that expresses the DNA repair protein, O(6)-methylguanine DNA methyltransferase (MGMT(P140K)) and selected in vivo with submyeloablative or myeloablative alkylator therapy. MATERIALS AND METHODS Lin(neg) BM was transduced at a low multiplicity-of-infection with the FV vector, MD9-P140K, which coexpresses MGMT(P140K) 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. RESULTS Following submyeloablative therapy, 55% of the mice expressed MGMT(P140K) in the BM. Proviral integration was observed in approximately 50% of committed BM-derived progenitors and analysis of proviral insertion sites indicated up to two 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, after delivery of myeloablative therapy to primary recipient mice, only 25% survived. Hematopoietic stem cells were transduced because BM cells from the surviving animals reconstituted secondary recipients with MGMT(P140K)-positive cells for 5 to 6 months. CONCLUSIONS In vivo selection of MD9-P140K-transduced BM cells was more efficient following submyeloablative than 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 acute toxicity associated with myeloablative therapy.

Preclinical activity of a novel multiple tyrosine kinase and aurora kinase inhibitor, ENMD-2076, against multiple myeloma.

ENMD‐2076 is a novel, orally‐active molecule that has been shown to have significant activity against aurora and multiple receptor tyrosine kinases. We investigated the activity of ENMD‐2076 against multiple myeloma (MM) cells in vitro and in vivo. ENMD‐2076 showed significant cytotoxicity against MM cell lines and primary cells, with minimal cytotoxicity to haematopoietic progenitors. ENMD‐2076 inhibited the phosphoinositide 3‐kinase/AKT pathway and downregulated survivin and X‐linked inhibitor of apoptosis as early as 6 h after treatment. With longer treatment (24–48 h), ENMD‐2076 also inhibited aurora A and B kinases, and induced G2/M cell cycle arrest. In non‐obese diabetic/severe combined immunodeficient mice implanted with H929 human plasmacytoma xenografts, oral treatment with ENMD‐2076 (50, 100, 200 mg/kg per day) resulted in a dose‐dependent inhibition of tumour growth. Immunohistochemical staining of excised tumours showed significant reduction in phospho‐Histone 3 (pH3), Ki‐67, and angiogenesis, and also a significant increase in cleaved caspase‐3 at all dose levels compared to tumours from vehicle‐treated mice. In addition, a significant reduction in p‐FGFR3 was observed on Western blot. ENMD‐2076 shows significant activity against MM cells in vitro and in vivo, and acts on several pathways important for myeloma cell growth and survival. These results provide preclinical rationale for clinical investigation of ENMD‐2076 in MM.

Virtual screening targeting the urokinase receptor, biochemical and cell-based studies, synthesis, pharmacokinetic characterization, and effect on breast tumor metastasis.

Virtual screening targeting the urokinase receptor (uPAR) led to (±)-3-(benzo[d][1,3]dioxol-5-yl)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-4-phenylbutan-1-amine 1 (IPR-1) and N-(3,5-dimethylphenyl)-1-(4-isopropylphenyl)-5-(piperidin-4-yl)-1H-pyrazole-4-carboxamide 3 (IPR-69). Synthesis of an analogue of 1, namely, 2 (IPR-9), and 3 led to breast MDA-MB-231 invasion, migration and adhesion assays with IC(50) near 30 μM. Both compounds blocked angiogenesis with IC(50) of 3 μM. Compounds 2 and 3 inhibited cell growth with IC(50) of 6 and 18 μM and induced apoptosis. Biochemical assays revealed leadlike properties for 3, but not 2. Compound 3 administered orally reached peak concentration of nearly 40 μM with a half-life of about 2 h. 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.

Granulocyte colony-stimulating factor prior to nonmyeloablative irradiation decreases murine host hematopoietic stem cell function and increases engraftment of donor marrow cells.

The use of nonmyeloablative conditioning prior to bone marrow transplantation is an important component of transplantation‐based therapies for nonmalignant blood diseases. In this study, treatment of recipient mice with granulocyte colony‐stimulating factor (G‐CSF) prior to low‐dose total body irradiation (LD‐TBI) enhanced long‐term engraftment of freshly isolated congenic marrow 1.5‐ to 2‐fold more than treatment with LD‐TBI alone. This combined regimen was also evaluated in a mouse model of X‐linked chronic granulomatous disease (X‐CGD), where neutrophils have a defective NADPH oxidase due to genetic deletion of the gp91phox subunit. Long‐term engraftment of male X‐CGD bone marrow cells cultured ex vivo for retroviral transduction of gp91phox was enhanced by ∼40% when female X‐CGD recipients were pretreated with G‐CSF prior to 300 cGy. These data confirm that sequential treatment with G‐CSF and LD‐TBI prior to transplantation increases long‐term engraftment of donor marrow, and they extend this approach to transplantation of murine donor marrow cultured ex vivo for gene transfer. Additional studies showed that the administration of G‐CSF prior to LD‐TBI did not alter early homing of donor marrow cells. However, the combined regimen significantly decreased the content of long‐term repopulating cells in recipient marrow compared with LD‐TBI alone, as assessed in competitive assays, which may contribute to the enhanced engraftment of donor marrow cells.

Enhanced homing and engraftment of fresh but not ex vivo cultured murine marrow cells in submyeloablated hosts following CD26 inhibition by Diprotin A

OBJECTIVE We recently reported that murine marrow cultured ex vivo for gamma-retrovirus transduction engrafts approximately 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. MATERIALS AND METHODS 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. RESULTS Homing of lin(-) cells cultured for gamma-retrovirus transduction was at least threefold less than that of fresh lin(-) cells 20 hours after transplantation into submyeloablated hosts. DipA treatment of fresh lin(-) cells resulted in at least twofold increased homing and engraftment in submyeloablated hosts. DipA treatment, however, did not significantly improve homing or engraftment of cells undergoing a 3-day culture protocol for gamma-retrovirus transduction in submyeloablated hosts. CXCR4 expression on lin(-) cells was significantly decreased following 3 days of culture; CXCR4 expression was not significantly altered following overnight culture. CONCLUSIONS 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.