AACRAmericanAssociationforCancerResearch
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`Lanestr ASSOCIATION
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`UAL MEETING OF THE
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`FOR CANCER RESEARCH
`
`rdAnnual Meeting
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`April 6-10, 2002 » San Francisco, California
`Volume 43 + March 2002
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`In joint sponsorship with the Keck School of Medicine of the University of Southern California
`
`The premier meeting for cancer research in the post-genomic era
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`# # e é ® . g f F ; Y
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`Featuring the latest developments in basic, translational, andclinical cancer research West-Ward Pharm.
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`aera
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`i y a A
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`Exhibit 1030
`Page 001
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`West-Ward Pharm.
`Exhibit 1030
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`AMERICAN ASSOCIATION FOR CANCER RESEARCH,INC.
`
`Officers
`
`President
`
`25 sss 0a nx oeewn ext ne ens ns: ssWaun Ki Hong
`
`President-Elect
`
`..0a oss 00 su rxauxean ens seSusan Band Horwitz
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`Treasurer 2... 2c eee eee eee Bayard D. Clarkson
`
`Past President ... 0... 00.0 c cee ce ee eeeTom Curran
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`Chief Executive Officer ... .
`
`. eee eect eeeMargaretFoti
`
`Board of Directors
`
`Until 2002
`MinaJ. Bissell
`Michael B. Kastan
`Edison T. Liu
`Frank J. Rauscher, HI
`
`Until 2004
`Until 2003
`Frederick R. Appelbaum
`Michaele C. Christian
`Ronald A. DePinho
`Lorraine J. Gudas
`Tyler E. Jacks
`William G. Nelson, V
`George F, Vande Woude
`Peter K. Vogt
`Barbara L. Weber
`Geoffrey M. Wahl
`
`
`Address inquiries to the Office of the American Association for Cancer Research, Inc. (AACR), Public Ledger Building, Suite 826, 150 S.
`Independence Mall West, Philadelphia, PA 19106-3483 [Telephone: (215) 440-9300; Fax: (215) 440-9313).
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`The Proceedings ofthe American Association for Cancer Research is printed for the AACR by CadmusProfessional Communications, Linthicum, MD
`21090-2908 and is included in the membership dues for active and associate members. Volume 43 of the Proceedings ofthe American Association for
`Cancer Research (ISSN 0197-016X) succeeds Volume 42 of the Proceedings of the American Association for Cancer Research. The Proceedings may be
`obtained at a price of $55.00 through registration at the Annual Meeting of the American Association for Cancer Research, April 6-10, 2002, or
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`The Proceedings ofthe American Association for Cancer Research is copyrighted © 2002 by the AACR.All rights reserved. Redistribution or resale of
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`Communications for the opinions expressed by the contributors herein.
`
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`AR.
`93rd Annual Meeting
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`West-Ward Pharm.
`Exhibit 1030
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`West-Ward Pharm.
`Exhibit 1030
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`differentiation of myeloid leukemia cell lines (NB4, HL-60 and U937). SS was
`identified in our recent studies as a potent inhibitor of PTPases. Herein, we
`sent data demonstrating that SS (250 ug/ml, 6 days) induced 87% of NB4
`cells to reducenitroblue tetrazolium (NBT), in comparison to the 90% induced by
`ATRA (1 »M, 6 days). SS-induced NB4 cell differentiation was confirmed by
`increased CD11b expression and associated with growth arrest at S phase and
`increased cell death. Our results showed further that SS-induced NB4 differen-
`tiation was irreversible and required continuous drug exposure for optimal induc-
`tion. Moreover, SS (400 g/ml, 6 days) induced 60% and 55% of NBT-positive
`cells in HL-60 and U937cell lines, which were augmented in the presence of
`GM-CSF (2 ng/ml) to levels (85% and 81%, respectively) comparable to those
`induced by ATRA. These results provide the first evidence of a differentiation
`induction activity of PTPase inhibitor SS in myeloid leukemia cell
`lines and
`gsuggest its potential therapeutic use in myeloid leukemia. Since SS induces
`differentiation via targeting PTPases, a mechanism distinct from that of ATRA,it
`may be particularly useful in AML cases unresponsive or developed resistance to
`ATRA.
`
`#356 NAD(P)H:quinone oxidoreductase (NQO1)-dependent and -indepen-
`dent cytotoxicity of potent quinone cdc25 phosphataseinhibitors. Yusheng
`Han, Hongmei Shen, Brian Carr, John S. Lazo, and Su-Shu Pan. University of
`Pittsburgh Cancer Institute, Pittsburgh, PA, and University of Pittsburgh, Pitts-
`burgh, PA.
`A vitamin K analogue, compound 5 (Cpd5, a thioethanol naphthoquinone),
`inhibits oncogenic Cdc25 phosphatases, and arrests cell cycle progression at
`both G1 and G2/M. Recently, we evaluated >10,000 compounds in the NCI
`chemical repository for in vitro inhibition against recombinant human Cdc?5B
`phosphatase and identified a quinone substructure in many of the active com-
`pounds. Bioreductive enzymesin cells, however, are known to reduce various
`quinones resulting in either detoxification or activation. Therefore, we used an
`isogenic set of human colon cancercell lines to evaluate the effect of NQO1 on
`the cytotoxic activity of Cpd5 and the two most potent phosphatase inhibitors
`from the repository: NSC 95397 (a bis-thioethanol naphthoquinone}) and NSC
`663284 (a quinolinedione). The two cancer cell lines used were HCT116, which
`has intermediate NQO1 activity, and its mitomycin C-resistant sub-line HCT116-
`R30A (R30A), which has minimum NQO1activity. Cell survival was measured by
`colony formation after 7 days drug exposure. Cell cycle arrest was evaluated by
`flow cytometry after 6 hr drug exposure. Cpd5 had an IC., of 2.2+0.3 uM for
`HCT116 and 0.23*0.05 uM for R30A,
`i.e. a 10-fold difference. Inclusion of
`dicoumarol (10 .M), an inhibitor of NQO1, decreased the IC, of Cpd5 for
`HCT116 to 0.24*0.04 4M, but had no effect on R30A. In contrast, HCT116 and
`R30A cells were equally inhibited by NSC 95375 with IC.,s of 1.4+0.3 uM and
`1.3+0.2 «M, respectively. Similarly, HCT116 and R30A cells were equally inhib-
`ited by NSC 663284 with IC5os of 2.40.3 »M and 2.6+0.5 pM, respectively.All
`three compounds blocked the two cell lines at the G2/M phase transition, con-
`sistent with cdc25inhibition. Cpd5 at 2.5 uM arrested R30A cells at G2/M but 7.5
`uM Cpd5 was needed to arrest HCT116 cells to a similar degree. NSC 95375 and
`663284 arrested cell cycle progression at G2/M of HCT116 and R30A cells
`similarly, and did so in a concentration-dependent manner between 2.5 and 7.5
`uM. Our data imply that NQO1 in HCT116 cells protected cells from the action of
`Cpd5, probably by the reduction of Cpd5 to a less active hydroxylquinone.In
`contrast, both NSC 95397 and 663284 displayed cytotoxicity that was indepen-
`dent of NQO1 levels. (Support: NCI CA61862 and CA78039)
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`EXPERIMENTAL/MOLECULAR THERAPEUTICS 3
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`reduced when rats were treated with acute (40% of control, p<0.00002) or
`chronic (50% of control, p<0.02) Cpd 5. Conclusions: Cpd 5 had significant
`inhibitory effect on growth of tumors and foci.
`
`#358 Bosentan, a novel endothelin-A and -B receptor antagonist inhibits
`proliferation of malignant melanomacells. Aleksandar Sekulic, Padma Suresh,
`Mark R.Pittelkow, and Svetomir N. Markovic. Mayo Foundation, Rochester, MN.
`Here we tested a feasibility of endothelin (ET) receptor blockade with a dual
`endothelin-A and -B receptor (ETR-A and ETR-B) antagonist, Bosentan, as a
`novel therapeutic approach for malignant melanoma. ETs are 21aa peptides
`primarily produced by endothelial cells and implicated in a variety of physiological
`functions. Binding of ET to ETR-A on vascular structures potently stimulates
`angiogenesis and,
`thus,
`likely plays an important role in growth of multiple
`cancers. Activation of ETR-Bs, amongotherthings, regulates melanocyte devel-
`opment and function. Wefirst examined patterns of ETR subtype expression on
`six established melanomacelllines using flow cytometry and immunocytochem-
`istry. Following this sections of primary and metastatic melanomatissues were
`evaluated for ETRA/ETRB expression by immunohistochemistry. To test func-
`tional effects Bosentan on melanomacell proliferation, six melanoma cell lines
`were subjected to standard 3H-thymidine incorporation assays in presence or
`absence of various concentrations of Bosentan. All examined melanomatissues
`(2 primary, 9 metastatic) express ETRB,albeit to different levels, whereas ETRA
`was expressed to low levels in only 3 metastatic tumors. In functional assays
`Bosentan inhibited proliferation of all examined cell lines with the IC50 ranging
`between 7 and 40 yg/mI. Ourresults suggest that malignant melanocytes express
`functional ETRs, and their treatment with Bosentan leads to significant growth
`inhibition. Concurrentinhibition of ETR-A and ETR-B jn vivo by low toxicity, orally
`available inhibitor Bosentan might therefore prove useful as a novel mode of
`anti-melanoma therapy through simultaneousinhibition of cancer cell growth and
`process of angiogenesis.
`
`#359 In vivo activity of RADOO1, an orally active rapamycin derivative, in
`experimental tumor models. Terence O'Reilly, Juliane Vaxelaire, Melanie Muller,
`Heinz-Herbert Fiebig, Marc Hattenberger, and Heidi A. Lane. Business Unit On-
`cology, Novartis Pharma AG, Basel, Switzerland, and Oncotest gmbH, Freiberg,
`Germany.
`RADOO01is a hydroxyethyl ether derivative of rapamycin thatis orally bioavail-
`able. RADOO1 has demonstrated in vitro anti-proliferative activity against a panel
`of human tumorlines. For in vivo testing, tumor-bearing nude mice were admin-
`istered RADO01 in a variety of doses and schedules. Tumors were established by
`transplantation of fragments generated from injection of cells, or by transplanta-
`tion of fragments from stabilized tumors originating from surgically removed
`human tumors. When administered once daily p.o., at doses ranging from 0.5-5.0
`mg/kg/day, RADOO1 was a potentinhibitor of tumor growth in 10 different
`xenograft models of human tumors(including pancreatic, colon, epidermoid, lung
`and melanoma).
`In general, RADOO1 was well tolerated and better tolerated in
`mouse xenograft models than standard cytotoxic agents (i.e. doxorubicin and
`5-fluorouracil), while possessing similar antitumor activity. Only one instanceofin
`vivo resistance has been observed (MAXF 401 mammary xenograft model),
`otherwise the activity of RADOO1 was generally inhibition of tumor growth (per-
`sistent regressionsin one tumorline, T/C values of 9 to 45 % in 8 tumor lines).
`Xenograft models sensitive to RADOO1 treatment included tumors exhibiting
`comparative resistance in vitro (KB-31 and HCT116). Persistent tumor regres-
`sions (41 %) were observed in a line displaying sensitivity to RADOO1 jn vitro
`(A549). Pharmacokinetic analyses, following a 5 mg/kg administration, indicated
`rapid uptake into plasma (Cmax 2513 ng/ml; Tmax 1h), but the time to Cmax was
`delayed in tumors (Cmax 102 ng/g; Tmax 2 h). Elimination from the tumor(t1/2,
`16 hr) was apparently slower than for plasma(t1/2, 7.5 hr). RADOO1 levels were
`abovethe IC50 of A549cells for a 72 h period. Interestingly, tumor RADOO1levels,
`following a single 5 mg/kg administration, never exceeded thein vitro antiprolif-
`erative IC50 for either KB-31 or HCT116 cells; despite the sensitivity of these lines
`in vivo. From these observations, and given the extreme sensitivity of endothelial
`cells to RADOO1, it is plausable that RADOO1 may not only act on tumorcells but
`mayalso affect angiogenesis. Taken together, these data support the application
`of RADOO1 as an antitumor agent.
`
`#357 Antitumor and anticarcinogenic action of Cpd 5: A new class of
`protein phosphatase inhibitor. Siddhartha Kar, Meifang Wang, Zhenggang Ren,
`Xiangbai Chen, and Brian |. Carr. University of Pittsburgh, Pittsburgh, PA.
`Background: We have chemically synthesized a newclass of inhibitors of dual
`Specificity phosphatases (DSP), which play an important role in cell cycle and
`signal transduction. Cpd 5 or 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoqui-
`none is one of the most potent. It inhibits DSPs (especially the Cdc25 family) in
`tissue culture cells and induces tyrosine phosphorylation of various DSP sub-
`strates, including Cdks and inhibits cell growth both in vitro and in vivo (JBC
`270:28304, 1995; Proc. AACR 39:224, 1998). Purpose: In this study we evaluated
`(a) the antitumor and(b) the anticarcinogenic activity of Cpd 5 for thefirst time.
`#360 Discovery of anticancer agents from sponge-associated fungi. Fred-
`erick A. Valeriote, Karen Tenney, Charles Grieshaber, Halina Pietraszkiewicz,
`Methods: (a) JM1 hepatomas were grown in 2 month old Fischer male rats by
`Subcutaneousinjection on the backor intra-portally in the liver. Rats were treated
`Akiko Amagata, Taro Amagata, Jeff Gautschi, Joseph Media, Joseph Stayanoff,
`Richard Wiegand, and Phil Crews. Henry Ford Health System, Detroit, Ml, and
`with Cpd 5 by intratumor, subcutaneous(nearby site), intramuscular(distant site),
`University of California Santa Cruz, Santa Cruz, CA.
`Or intraperitonealinjection, either as a single high acute dose or chronically as
`Several low doses.(b) Rats were injected intraperitoneally with a single doseof the
`We have evaluated 1,112 extracts (from 660 sponge-associated fungi) for
`assessment of potential anticancer activity. Both broth and mycelia extracts were
`Carcinogen N-Nitrosodiethylamine (DEN). Immunostainedliver sections for gluta-
`assayed in most cases. Each sample was assayed jin vitro against up to8cell
`thione-S-transferase-pi (GST-pi) detected pre-neoplastic foci after 3 weeks. Cpd
`types (murine and human) in a disk diffusion/ clonogenic assay. From these
`5 was injected subcutaneously or intraperitoneally two weeks after DEN as a
`Single high acute dose or chronically as several low doses. Results: (a) Cpd 5 had
`results, the samples were assigned into one of 4 categories: Inactive (79% of the
`Significant inhibitory effect on both intrahepatic (14% of control, p<0.00000008)
`extracts), Equally active across cell types (16% of the extracts), Equally active and
`and subcutaneous (33% of control, p<0.00008) tumor growth and also had
`potent (9 extracts or 1%), and Solid tumor selective (42 extracts or 3.8%). The
`Significant inhibitory effect when injected intramuscularly ata site distant from the
`equally active and potent category is studied further since solid tumor selective
`tumor(50% of control, p=0.002). There was no significant difference between the
`compounds might exist in the extract but be concealed by one or more potent,
`cytotoxic compounds. Further, a novel, potent compound could form the basis
`effects after acute or chronic injections. However, toxicity was much lower with
`for analog synthesis in an attempt to develop an active anticancer agent. One
`Chronic treatment. (b) The number of enzyme altered foci was also significantly
`
`Proceedings of the American Association for Cancer Research ¢ Volume 43 ® March 2002
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`71
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`This material may be protected by Copyright law (Title 17 U.S. Code)
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