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`DR. REDDY’S LABS., INC. EX. 1058 PAGE 1
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`DR. REDDY’S LABS., INC. EX. 1058 PAGE 1
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`MOLECULAR PHARMACOLOGY & DRUG RESISTANCE II
`
`in nine of twenty-five AML patient samples. There was a strong correlation
`colony mhibition,
`betweeri DTrrsIL3]og celt kill and blast high affinity IL3 receptor density (p = 0 0044) and
`no correlation between low affinity receptor density and log cell loll. These results suggest
`blasts to DT„,It3
`in the sensrtivity of patrents'eukenuc
`that at least one factor important
`rs rts abihty to bind to the cell surface DTiuIL3 shows toxicity to patient
`leukemic blasts
`as well as cell lines and suggests
`the drug merits further preclinicat development,
`that
`In
`the presence of high affinity IL3 receptors on patients'lasts may predict clmica]
`addition,
`to DTrrs]L3 in chnical
`tnals
`responsiveness
`
`F
`
`1Cl
`
`P
`
`0
`
`1000
`1250
`750
`500
`250
`IL3 High Aftlnity Receptor Density
`
`I
`
`1500
`
`Mu]ter*,'avid
`
`Hideshima*,'harminder
`
`363-II
`1313
`Poster Board ¹-Session:
`Abstract¹
`THALIDOMIDE (THAL) AND ITS ANALOGS OVERCOME
`DRUG RESISTANCE OF HUMAN MULTIPLE MYELOMA (MM)
`CELLS TO CONVENTIONAL THERAPY. Teru
`Shima*,'aje Noopur*,'aith E,
`Chauhau*,'oshihito
`Davies*,'u-Tzu Tai*,'teven P. Treou,'oris K. Liu*,'obert L.
`Schlossman,'aul G. Richardson,'eepak Gupta*,'eorge W.
`I. Stirling*, Kenneth C. Anderson.'Adult
`Oncology, Dana-
`Farber Cancer
`and Harvard Medical School, Boston, MA;
`Institute
`'Ce]gene Corporation, Warren, NJ.
`Although That was initially used to treat MM due to its known anti-angiogenic
`effects,
`the mechanisms of its anti-MM activity are undefined.
`In this study, we assessed the activity
`of That, as well as its analogs
`the Selected Cytokine
`(SelCIDs) and
`Inhibitory Drugs
`Drugs (IMiDs), against human MM cells. Like That, both Se]CIDs and
`Immunomodulatory
`IMiDs inhibit TNFa; SelCIDs are phosphodtesterase
`4 inhibitors
`(PDE41) which do not
`enhance T cell activation, whereas
`IMiDs are not PDE4I and stimulate T cell proliferation.
`That (100I(M) and IMrDs (0.1-1.0I(M), but not Se]CIDs, rhrectly induce aPoPtosis or Gl
`growth arrest m MM cell lines and patient MM cells which are resistant
`to melphalan,
`(Dex). That and the IMiDs also enhance
`doxorubicin,
`the anti-MM
`and dexamethasone
`activity of Dex and are partially
`by interleukin-6.
`IMiDs induced apoptosis
`inhibited
`assocrated with decreased p21 expression in MM.]S cells (wild type p53) versus Gl growth
`arrest associated with increased p21 in Hs-Sultan and patient MM cells (mutant p53). As
`for Dex and in contrast
`to gamma irradiation and Fas induced cell death, apoptotic signaling
`triggered by That and the IMiDs in MM cells is associated with activation of related adhesion
`focal tyrosine kinase (RAFTK). These studies therefore demonstrate direct activity of That
`and IMiDs on MM cells resistant
`to conventional
`therapy. Thai and the IivhDs therefore
`represent a new treatment paradigm targeting both the tumor cell and the microenvironment
`to overcome classical drug resistance and achieve improved outcome in MM.
`
`364-II
`1314
`Poster Board ¹-Session:
`Abstract¹
`GROWTH OF ACUTE LYMPHOBLASTIC LEUKEMIA CELLS
`IS SUPPRESSED BY jI-I-ADRENERGIC AGONISTS AND THE
`NOVEL WATER-SOLUBLE FORSKOLIN NKH477. Ryosuke
`J. Kato.'Division
`Ogawa,'rtem Bugayenko*,'regory
`of Pediatric
`Johns Hopkins University School of Medicfne, Baltimore,
`Hematology,
`'First Department
`of Internal Medicine, University
`of
`MD, USA;
`School of Medicine,
`Occupational
`and Environmental
`Health
`Japan.
`Kitakyushu,
`that elevation of rntracellular
`evidence indicates
`Substantial
`cyclic AMP suppresses
`in thymocytes. Data from our lab and others indicate that
`growth and induces apoptosis
`cultured T-ce]1 and B precursor acute lymph oblasnc leukemia cells exhibit similar sensitivity
`to elevation of cAMP by phosphodiesterase
`a survey of
`inhibitors. We have undertaken
`cyclase activators
`that might provide together wrth phosphodiesterase
`inhibitors
`adenylyl
`elevation of cAMP We have previously
`reported such a synergy of the PDE4
`synergistic
`forskolin. A congener of
`rolipram with the model adenylyl
`inhibitor
`cyclase activator
`the water-soluble
`compound NKH477,
`forskolin,
`is utilized in Japan as an inotropic agent
`for refractory heart failure. Using a modified MTT assay on the CEM-CCRF model of T-cell
`cell growth with 50% mhibr tron (IC50) provided
`ALL, we find that
`this drug suppresses
`by 40 micromolar NKH477. The addition of the PDFA inhibitor
`rohpram to NKH477
`of cell growth. 20 micromolar NKH477 reverses
`provides
`synergistic
`suppression
`glucocorticoid resistance in these cells, with a two to three log reduction m the dexamethasone
`IC50. We also mvestigated
`potential of I)-adrenergic
`the growth suppressive
`agonists,
`which also can induce activation of adenylyl cyclase. We find that the ]I-adrenergic agon(st
`and II-]-adrenergic
`rsoproterenol
`potently elicit growth suppression,
`agon(st dobutanune
`
`304a
`
`360-II
`1310
`Poster Board ¹-Sess]or(:
`Abstract¹
`DERECULATION OF C-MYC GENE EXPRESSION BY THE
`HUMAN IgH 3'NHANCER IN BURKITT'S LYMPHOMA. Lu
`Zhang~, Lina Somsouk*, Linda M. Boxer. Division of Hematology,
`Stanford University,
`Stanford, CA, USA.
`The expression of the c-myc gene rs deregulated
`heavy chain
`by the immunoglobu]in
`(IgH) 3'nhancers
`in Burkrtt's
`cells with the t(8; 14) translocation.
`Several
`lymphoma
`studies have exaniined the acnvity of the murine IgH enhancers with the c-myc promoter. To
`study the mechanism of activation of c-myc by the human IgH enhancers, we cloned the
`human B-cell specific hypersensitive
`sites (HHS]2, HHS3 and HHS4) from the lgH enhancer
`C(r I region Usmg a reporter system, we tested the activation of the c-myc promoter by these
`HHS regions The most 3'nhancer HHS4 showed a B-cell stage specific pattern. It increased
`c-myc expressron 7.8-fold m both onentatrons
`in Raji Burkrtt's
`cells but
`less
`lymphoma
`for most of the activity of
`than I 7-fold ni DHL-9 cells An NF-kB site was responsible
`HHS4. Human HS3 in both orientations
`increased c-myc expression slightly (2.4-fold) in
`Rap and 1.4-fold in DHL-9 cells Deletion analysis
`identified two regtons that were active.
`Several different HHS]2 forms have been cloned that differ by the number of copies of a 53-
`bp mo(if which contarned a putative NF-kB bindrng site. However, constructs that contained
`different copy numbers of this repeat (I, 2 and 3 copies) showed no significant difference in
`activity in both Rap and DHL-9 cells To further define the regulatory elements within the
`region, deletion analysis was performed. A region 3'f the 53bp repeat
`HHS]2 enhancer
`of the c-myc promoter. Deletion of this 30bp
`showed a posinve effect on tianscnption
`level. UV cross-linhng
`region decreased the activity to the basal
`studies showed that
`two
`(40 kDa and 80 kDa) bound to the region. EMSA competition studies showed that
`proteins
`STAT4 rs a potential candidate for binding to ttus srte. 3'eletions also identified two other
`active regions m HHS]2 Strong synergistic mteractions were observed among the
`enhancers HS],2,3, and 4 with the c-myc promoter The HHS124 combination yielded a 26-
`a ]3-fold and HHS]23 combmation
`fold, HHS34 combination
`a 7-fold induction in c-myc
`expression m Raji cells. The combination of all four enhancers
`showed the highest activity
`with a 42-fold induction of c-myc promoter activity in Raji cells.
`
`361-1I
`1311
`Poster Board ¹-Sessionf
`Abstract¹
`DEREGULATION OF NOTCH2 SIGNALLING IN B-CLL. Rainer
`Hubmanu",'osef
`Schwarzmeier*," Medhat Shehata*,' Martin
`Hi]garth",'udolf Berger*,']aus Lechner.'Internal Medicine
`I,
`'L. Boltzmann
`Inst. for Cytokine
`University of Vienna, Vienna, Austria;
`Ites., University
`of Vienna, Vienna, Austria.
`Members of the Notch gene family encode transmembrane
`that control
`receptors
`and apoptotic programs of many types of progenitor
`cells includmg
`differentiation
`precursors. Activation of Notch by its ligand causes cleavage and
`hematopoetrc
`translocation of the intracellular
`to the nucleus, where it activates
`domain (NotchIC)
`the
`factor CBFI. The aim of this study was to elumdate the mechanisms
`leading to
`transcription
`the overexpression of the CD23a isoform in peripheral blood B-cell chronic lymphocytic
`to be related to B-CLL cell viability. By
`(B-CLL) cells which is supposed
`leukemia
`electrophoretic mobility stuft assays (EMSA), we identified a transcription
`factor complex
`(Cl) which binds to one known and four newly identified putative CBF] sites in the CD23a
`by the fact, that in B-
`proximal promoter. The significance of this complex was highlighted
`cell samples the intensity of Cl correlated with their respective levels of CD23a expression.
`for CBF]
`usmg Epstein Ban virus {EBV)infected BL41 cells as a model
`Furthermore,
`mediated CD23 expression, Cl was found to be EBV inducible. Since CBF] is the nuclear
`target of Notch signahng, we investigated the expression pattern of different members of the
`in B-CLL cells.
`Notch gene family by RT-PCR and found that Notch2IC is overexpressed
`active Notch is known to lock cells into an immature
`to
`state and appears
`Constitutively
`inhibit apoptosis rn certain leukemic cell lines. Therefore, deregulation of Notch2 signaling
`would be consrstant with the nature of B-CLL cells indicating
`that Notch2 might play a
`of this disease
`role m ihe pathogenesrs
`pivotal
`
`MOLECULAR PHARMACOLOGY & DRUG RESISTANCE II
`
`362-II
`1312
`Poster Board ¹-Session:
`Abstract¹
`HIGH AFFiINITY INTERLEUKIN-3 RECEPTOR EXPRESSION ON
`BLASTS FROM PATIENTS WITH ACUTE MYELOGENOUS
`LEUKEMIA CORRELATES WITH CYTOTOXICITY OF A
`DIPHTHERIA TOXIN/IL-3 FUSION PROTEIN. Richard L.
`Jason Ramagev', Barbara K]eius, Gregory L. Kucera"',
`Alexander",
`Michea] A. Ca]igiuri, Clara D. Bloomfield, Arthur E. Franke]. Cancer
`/Biology, Wake Forest University School of Medicine, Winston-Salem,
`Cancer
`St. Louis, MO, USA; Comprehensive
`NC, USA; Pharmacra,
`Center, Ohio State University, Columbus, OH, USA.
`We have constructed a fusion protein (DT„„IL3)in which we fused interleukin-3
`(IL3)
`to the catalytic and tianslocatron domains of diphthena
`(oxin (DT) (Leukemia 14:576-585,
`2000 and Protein Engmeenng
`13:575-581,2000) Previously, we demonstrated
`that
`DT„„,IL3was toxic to a fraction of myeloid leukenua
`cell lines (Biocon]ugate Chemistry
`the sensiuvity of patient
`11.564-568,2000). Next, we wanted to determine
`leukenuc blasts
`to DTrrr]L3 and factors (hat may predict cytotoxrcrty To this end, IL3 receptor density and
`DT„,IL3sensitivity of acute myeloid leukemia (AML)-colony formmg cells (CFC) were
`measured ui leukenuc blasts from twenty-five AML patients. The blasts had high affinity
`(Kd = 16+ 15 pM n = 25) IL3 receptor densities that ranged from 0 to 1220 receptors per cell
`and low affinny (Kd = 7060 + 6938 pM n = 25) receptor densities that ranged from 263 to
`18250 receptors per cell We observed greater than one log cell kill, as measured by percent
`
`DR. REDDY’S LABS., INC. EX. 1058 PAGE 2
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