Shantaram S. Joshi, Ph.D.

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1 Shantaram S. Joshi, Ph.D. Functional Genomics of Leukemia Dendritic Cell Based Therapy for Cancer Dept. of Genetics, Cell Biology and Anatomy University of Nebraska Medical Center Omaha, Nebraska Phone: /4560, Fax: ,

2 Research Summary Overall Goals: The long-term goal of this laboratory is to improve therapy for human B lymphocyte malignancies. Where feasible, to apply the same approach to other malignancies, such as breast cancer and also cancers of the nervous system. This is a translational research laboratory as described below. Project I: Improved Therapy for Chronic Lymphocytic Leukemia (CLL): Gene Targeting Approach using Functional Genomics CLL is a common leukemia of the older population in Western countries. Resistance to therapy due to alterations in apoptosis is a major problem in clinical management of CLL. Using DNA microarray analysis, the differentially expressed genes in therapy resistant CLL cells from patients, are being identified. Selected differentially expressed genes were targeted to revert their altered expression which resulted in making resistant CLL cells significantly more susceptible to therapy. Collaborators: Collaborators in this study are Drs. Phillip Bierman, M.D. Greg Bociek, M.D. John (Wing) Chan, M.D., Steve Pavletic, M.D.; and James Lynch, Ph.D. Potential Impact: This is truly a translational research project where patient cells are analyzed to improve the therapy for CLL. One of the differentially expressed genes, Bcl-2, identified by us and others is being targeted in a multi center Phase II Clinical trial including UNMC sponsored by Genta Inc and NCI. Several candidate genes for targeting are being reviewed.

3 Project II: Use of Umbilical Cord Blood (UCBC) for Cellular Therapy Against Cancer: We have shown that human umbilical cord blood contain precursors of both antigen non-specific and antigen specific cytotoxic effector cells against leukemia/lymphoma and breast cancer. IL-2 activated killer cells and Her2/neu specific CTLs showed a significant antitumor cytotoxicity in vitro and in vivo against human leukemia/lymphoma and breast cancer. Collaborators: Drs. Anne Kessinger, M.D., Charles Kuszunski, Ph.D., Samuel Pirruccello, M.D., and Stefano Tarantolo, M.D. Potential Impact: This is another translational research project in which the laboratory results have lead to patient treatment. Both in animal models and in patient we have shown the therapeutic effects of UCBC. A PhaseI /II clinical trial is being planned by Drs. Tarantolo and Haupke. A B Legend: A: Cord Blood Derived DC Ultrastructure; B: Antigen presentation by DC to T Lymphocytes Project III: Dendritic Cell Based Therapy for Mantle Cell Lymphoma and Other Cancers. The main objective of this project is to stimulate DCs to initiate antitumor response using two different approaches. In one approach DCs derived from human peripheral blood or umbilical cord blood mononuclear cells will be primed with tumor specific antigens such as Her2/neu and N-Myc. Such antigen primed DCs are being used to generate CTLs against specific tumor cells. In the second approach, which is being done for mantle cell lymphoma (MCL), DCs will be generated from mononuclear cells from MCL patients and fused with autologous lymphoma cells. Such DC-MCL hybrids will then be used as vaccine against Mantle Cell Lymphoma. Collaborators: Drs. Anne Kessinger, M.D., Stefano Tarantolo, M.D.;and Julie Vose, M.D. Potential Impact: If successful in preclinical studies, this translational research will be used in a clinical setting to treat mantle cell lymphoma patients.

4 Funding Sources 1) Elsa U. Pardee Foundation, Midland MI; 2) I Berlex Laboratories, Richmond, CA ; 3) Susan G. Komen Foundation, Dallas, TX; 4) Nebraska State Department of Health, LB506 Funds, Lincoln, NE and 5) National Institutes of Health, Bethesda, MD. Publications from Bagchi D, Joshi SS, Bagchi M, Balamoori J, Benner EJ, Kuszynski CA, Stoh SJ. Cadmium and chromium induced oxidative stress, DNA damage and apoptotic cell death in cultured human chronic myelogenous leukemic K562 cells, promyelocytic HL-60 cells and normal human peripheral blood mononuclear cells. J. Biochemical and Molecular Toxicology 14: 33-41, Liu C, Ferdinandi ES, Ely G, Joshi SS. Virulizin-2γ, a novel immunotherapeutic agent in the treatment of human pancreatic cancer xenografts. Int. J. Oncology, 16: , Mata JE, Jackson JD, Joshi SS, Tracewell WG, Pirruccello SJ, Murphy BO, Bishop MR, Iversen PL. In vivo effects of sixbase phosphorothioate oligonucleotide with anticancer and hematopoietic activities in Swine. J. Hematotherapy and Stem Cell Research, 9: , Joshi SS, Miller K, Kessinger A. Functional and phenotypic properties of peripheral blood stem cell transplantation following mobilization with erythropoietin + G-CSF in Cancer patients. Cytotherapy 2: 15-24, Bagchi D, Bagchi M, Stohs SJ, Das DK, Ray SD, Kuszynski CA, Joshi SS, Preuss HG. Free radicals and grape seed proanthocyanidin extract: Importance in human health and disease prevention. Toxicology 148: , Joshi SS, Kuszynski CA, Benner EJ, Bagchi M, Bagchi D. Chemopreventive effects of grape seed proanthocyanidin extract on Chang liver cells. Toxicology 155: 83-90, 2000 Joshi SS, Kuszynski CA, Tarantolo SR, Kessinger A. Antitumor therapeutic potential of activated human umbilical cord blood cells against leukemia and breast cancer. Clinical Cancer Research 6: , Sharp JG, Kessinger A, Lynch JC, Pavletic ZS, Joshi SS. Blood stem cell transplantation: Factors influencing immunological reconstitution. J. Hematotherapy and Stem Cell Research 7: , Vu UE, Wang X, Pavletic ZS, Joshi SS Increased cytotoxicity against B-CLL by cellular manipulation: Potentials for therapeutics Leukemia Lymphoma, 39: , 2000.

5 Joshi SS, Kuszynski CA, Das DK, Bagchi D. Chemopreventive effects of grape seed proanthocyanidin extract: Molecular mechanism of action. Current Pharmaceutical Biotechnology 2:, , Bagchi M, Joshi SS, Kuszynski CA, YE X, Stoh SJ and Bagchi D. Protective effects of a novel grape seed proanthocyanidin extract on smokeless tobacco induced oxidative stress and modulation of bcl-2, p53 and c-myc genes. Free Radical Research 35: , Joshi SS, Lynch JC, Pavletic ZS, Tarantolo SR, Kessinger A, Bishop MR. Decreased immune functions of blood cells following mobilization with G-CSF: Association with donor characteristics. Blood, 98: , Sharp JG, Bishop MR, Copple TC, Greiner TC, Iversen PL, Jackson JD, Joshi SS, Benner EJ, Manner SL, Rao AK, Vose JM. Oligonucleotide enhanced cytotoxicity of idarubicin for lymphoma cells. Leukemia and Lymphoma. 42: , Joshi SS, Lovgren TL, Wang P, Vu UE, Joshi BJ and Dave HPG. Kinetics of generation of functional dendritic cells from human cord blood mononuclear cells using defined medium. J. Hematotherapy and Stem Cell Research 11: , Wang P, Z.S. Pavletic and Joshi SS, Increased apoptosis of B-chronic lymphocytic leukemia cells with fludarabine by downregulating cyclin-d3 expression. Leukemia and Lymphoma 43: , Lovgren TL, Cody E, Lovgren TL, Wang P, Kuszynski CA, Tarantolo S, Joshi SS Combination of Interleukins and colony stimulating factors enhance cytotoxicity of umbilical cord blood cells against human breast cancer. In Vivo, 16: , Murphy BO, Joshi SS, Reed B, Kessinger A, Sharp JG. Animal model for bone marow metastasis of breast cancer. Clin. Exptl. Metastasis, 19: , Wang P. Munger CM., Pirruccello SJ Joshi SS. Cytotoxicity of Cord Blood Derived Her2/neu specific CTLs aganst human breast cancer in vitro and in vivo. Breast Cancer Res and Treatment (2003 in revision). Cluster analysis of CLL DNA Microarray Results

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