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`West-Ward Exhibit 1095
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`Yu 2001 - Page 001
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`West-Ward Exhibit 1095
`Yu 2001 - Page 001
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`EXPERIMENTAL/MOLECULAR THERAPEUTICS 41
`
`IGF' 1 stimulated phosphorylation of Erk-1 and Erk-2. The cells were exposed for
`2 hours to graded concentrations of PD 98059 (0.1 to 15.0 microM ) and stimu(cid:173)
`lated for 5 minutes with IGF-1 (10 ng I mL).and the phosphorylation levels of Erk-1
`and Erk-2 were detected by Western blot using phospho-Erk 1 I 2 ( p42/44 )
`antibody. At 1microM PD 98059 completely suppressed the activation of Erk-1
`and Erk-2. Further, IGF+stimulated ERK-1/2 phosphorylation was inhibited for
`up to 6 days by PD98059 (1 to 15 microM). Similar results were obtained with
`U0126, another MEK1 inhibitor. Of interest, IGF-1 protected against rapamycin in
`the presence of U0126 or PD98059. These results indicate that IGF+mediated
`rescue from rapamycincinduced apoptosis, and growth inhibition is independent
`oi both mTOR and ERK-1/2. To elucidate the pathway(s) involved we have started
`to analyze changes in transcription induced by IGF-1 in the presence of rapamycin
`plus PD98059. Initial results using an Affymetrix HG-U95A Gene Chip (12,559
`human genes), indicate that expression of relatively few genes change following
`1 hr IGF-1 stimulation (0.65% > 2-fold: 19 increased, 65 decreased). Supported
`by CA23099, CA77776 and by ALSAC.
`
`#4303 The PKC Activator Bryostatin 1 Pote"tiates Ara-C-lnduced Apo(cid:173)
`ptosis in Human Leukemia Cells Overexpressing Bel-XI by Promoting Cyto(cid:173)
`chrome· ·C Release. Zhiliang Wang, Shujie Jenny Wang, and Steven Grant.
`Medical College of Virginia, Richmond, VA.
`We pr~viously reported that in U93l cells, the macrocyclic lactone bryostatin 1
`(Bry) · partfally overcame resistance· to ara-C-mediated apoptosis conferred by
`overexpression of Bcl-2·(Wang et al., Mol Pharm 52:1000, 1997). The purpose of
`the present studies was to extend these findings to the anti-apoptotic protein
`Bcl-xL, the cytoprotective effects of which have been shown to differ from those
`ofbcl-2, and to explore mechanisms by which enhanced apoptosis might occur.
`Enforced expression of bcl-xL markedly reduced ara-C (1 µ.M; 6 hr)-induced
`cytochrome c release, loss of A 'I'm, procaspase-3 cleavage, and PARP degra(cid:173)
`dation. Subsequent exposure of U937 /Bcl-xL cells to bry (1 O nM; 18 hr) partially
`restored ara-C-induced cytochrome c release, loss of A 'I'm, caspase-3 and -9
`activity·ao_d apoptosis. While the caspase inhibitor ZVAD-fmk blocked ara-C/Bry(cid:173)
`induced apoptosis and loss of A 'I'm in U937 /Bcl-xL cells, it failed to oppose
`cytochrome c release, suggesting the latter represents a primary event in poten(cid:173)
`tiation of apoptosis. Bry did not alter levels of apf-1, nor did it increase the amount
`of Bel-XL co-immunoprecipitating with this protein. Exposure of U937/Bcl-xL(cid:173)
`overexpressing cells to Bry led to alterations in the mobility of Bcl-xL following
`PAGE analysis, manifest by a broadening of the ectopically expressed Bcl-xL
`protein band. Lastly, subsequent exposure of U937/Bcl-xL cells to ara-C-> Bry
`re.duced colony formation to levels observed in wild-type cells exposed to ara-C
`alone .. These findings indicate that Bry partially restores ara-C sensitivity in cells
`ectopically expressing Bcl-xL through a mechanism that involves potentiation of
`cytochrome c release.
`
`#4304
`In-Vitro Effect of the Epidermal Growth Factor Receptor Inhibitor
`PKI 166 on Human Lung Cancer Cell Lines. Kil Dong Kim, Rachel Zuei T. Tsang,
`Waun Ki· Hong, and Roy S. Herbst. The University of Texas M. D. Anderson Cancer
`Center, Houston, TX.
`Epidermal Growth factor receptor (EGFR) is a member of a family of four growth
`factor receptors [EGFR(HER1 or ErbB1), ErbB2 (HER2/neu), ErbB3 (HER3), and
`ErbB4 (HER4)]. These receptors are large proteins (-178KD) which reside in the
`cell membrane, each with a specific external ligand binding domain. High EGFR
`expression has been observed in 75% of lung cancer patients and hence repre(cid:173)
`sents an excellent target for therapy. We investigated EGFR expression of the
`Non-small cell lung cancer (NSCLC) cell lines, H226 (squamous), H322 (Bron(cid:173)
`choalveolar,BAC), A549 (adenocarcinoma), H1299 (large cell), H358 (BAG.), H460
`(large cell.), H596 (adenosquamous), H522 (adenocarcinoma.), PC14PE6 (adeno(cid:173)
`carcinoma), H1792 (adenocarcinoma H292 (mucoepidermoid), H157 (squamous)
`and DMS-4C(adenot:arcinoma.) using western blot analysis. The H226, H322,
`H358, A549, H292 and H157 cell lines demonstrated increased EGFR expression.
`H226 had the strongest expression of EGFR (+++),while the others had less but
`measurable expression: H157 (++), H292 (++), H322 (++), H358 (+)and A549
`(++).·We next investigated these highs expressing cell lines for activated EGFR
`activity using an anti-phosphotyrosine antibody with and without exogenous EGF
`stimulation and studied the ability of PKI 166, a small molecule, oral EGFR
`tyrosine kinase inhibitor to block this process. The concentration of PKI 166 used
`ranged from 0 to 2.5µ.M. In the cell lines absent EGF stimulation, there was
`minimal phosphorylated tyrosine kinase activity observed at baseline. In the cell
`lines with EGF stimulation, the signal intensity of phosphorylated tyrosine kinase
`was enhanced and decreased in the immunoprecipitation :;;tudy with the addition
`of PKl-166. The cell lines were differentially inhibited by PKl-166: H226 (-), H157
`(-).H292 (-), H322 (.,.), H358 (-), A549 (-).In most cases, this inhibition correlated
`with the baseline level of EGFR. These results demonstrate the potential use of
`PK1-166 in in-vivo studies and therapeutic animal models are currently underway.
`
`#4305 Deregulated P13k/AKT/TOR Pathway in PTEN-Deficient Tumor
`Cells Correlates with an Increased Growth Inhibition Sensitivity to a TOR
`Kinase Inhibitor CCl-779. K. Yu, W. Zhang, J. Lucas, L. Toral-Barza, R.
`Peterson, J. Skotnicki, P. Frost, and J. Gibbons. Department of Oncologyllmmu(cid:173)
`noinflammation, Wyeth-Ayerst Research, Pearl River, NY.
`Deregulation of the P13K/ AKT /TOR signaling pathway is widely believed to play
`a major role in human cancer. Genetic deletion or mutation of the PTEN/MMAC1
`
`tumor suppressor, a dual specificity phosphatase and a critical negative regulator
`of this pathway, is estimated to occur in as many as 50% of all solid human
`tumors. GCl-779, a macrolide inhibitor of the TOR kinase, is presently undergoing
`clinical cancer trials. The mode of action by CCl-779 suggests that the drug will
`be an effective therapy ·for human tumors, and it may demonstrate an even
`greater activity against tumor cells with elevated AKT/TOR signaling caused by
`cellular alterations, including the loss of PTEN tumor suppressor. We have con(cid:173)
`ducted a survey of the levels of the phophorylated (active) AKT in a panel of brain,
`prostate, breast and colon tumor cell lines. The phospho-AKT status in these cells
`were further compared with their growth inhibition sensitivity to CCl-779. We
`found that in serveral tumor types, loss of PTEN tumor suppressor and/or acti(cid:173)
`vation of AKT correlated directly with an increased susceptibility to CCl-779. In
`most cases, tumor cells that are resistant to CCl-779 are found to have low or a
`moderate level of activated AKT. These in vitro cell culture observatons have been
`further supported by the activity of CCl-779 against the growth of human tumors
`implanted in nude mice. Furthermore, several cell lines that contain a moderate
`level of activated AKT are also sensitive to CCl-779. Our data indicate that an
`increased phosphorylation .of AKT renders cells more sensitive to the inhibition,
`but it is not a requirement for the inhibition by the drug.
`
`#4306 ZD1839 ('lressa'), an EGFR Tyrosine Kinase Inhibitor, Potentiates
`Non-Mhc Restricted Cytotoxlcity in Human Cancer Cell Lines. Alfredo Bud(cid:173)
`illon, 'R: ~uarrasi, E. Di Gennaro, F. Bruzzese, S. Errico, G. Pirozzi, M. Caraglia, A.
`Avallone, P. F. Tassone!, M. L. Lombardi, F. Caponigro, S. Venuta, and P.
`Tagliaferri. Magna Graecia University, Catanzaro, Italy, and National Cancer lnsti(cid:173)
`tl.lte - G. Pascale, Napoli, Italy.
`Disregulated expression or function of the growth factor receptors of the erbB2
`family is a common finding in several human tumors. The growth factor mediated(cid:173)
`signalling has beeri considered to promote cell proliferation and also to behave as
`a survival, proangiogenic and antiapoptotic pathway. Overexpression and func(cid:173)
`tional alterations of erbB family members has also been correlated to resistance
`of'lumor cells to non-MHC restricted cytotoxicity. We have previously shown an
`increased resistance to Lymphokine activated Killer (LAK)-mediated cytolysis in
`immortalized MCF-1 O breast cells overexpressing erbB2 (P. Tagliaferri et al CCR,
`1996). In addition, using a cold target competition assay, we have demonstrated
`that resistance to LAK is not due to altered effector/target binding, suggesting
`that the activation of growth factor signalling induces resistance to immune
`cytolysis acting downstream to the lymphocyte recognition of tumor cells. On
`these bases, we have investigated whether ZD1839 ('lressa'), an orally active,
`selective EGFR tyrosine kinase inhibitor (EGFR-TKI), may increase the sensitivity
`of tumor cell lines to non-MHC restricted cytotoxicity. We have found that
`untreated HNSCC and Melanoma cells expressing high levels of EGF-R and of
`other erbB receptors were almost completely .resistant to immune-cell killing but
`showed increased sensitivity to LAK-mediated cytotoxicity, as demonstrated by
`LDH-specific release assay, after ZD1839 treatment. In particular, we have dem(cid:173)
`onstrated a dose dependency (treatment range 0.1-5 µ.M ZD1839) which was
`already evident after 6 h treatment and decreased after 48 h. Induction of
`sensitivity to activated lymphocytes occurred in parallel to downregulation of
`ERK-1/ERK-2 activity and could be detected when cells had not yet lost full
`viability. These results suggest that EGFR mediated signalling could represent a
`useful cellular target for overcoming tumor cell resistance to activated lympho(cid:173)
`cytes and provide a potential rationale for the clinical use of the EGFR tyrosine
`kinase inhibitor ZD1839 ('lressa') in combination with immunostimulating cyto(cid:173)
`kines. 'lressa' is a trade mark of the AstraZeneca group of companies.
`
`#4307 Epidermal Growth Factor Receptor Tyrosine Kinase Inhibition by
`ZD1839 ('lressa') Blocks Oestrogen-Stimulated Proliferation and Progester(cid:173)
`one Receptor Expression in Human Breast Epithelium. Kai C. Chan, W. Fiona
`Knox, R. Dobson, C. S. Potten, and N. J. Bundred. University Hospital of South
`Mancihester, Dept. of Surgery, Manchester, UK, and University Hospital of South
`Manchester, Dept. of Surgery, Manchester, UK.
`Normal breast epithelium commonly expresses epidermal growth factor recep(cid:173)
`tor (EG.FR) and oestrogen receptor. Several in vitro studies suggest that oestro(cid:173)
`gen stimulates proliferation by utilising the EGFR pathway. We sought to deter(cid:173)
`tyrosine kinase
`inhibitor, decreases
`mine if ZD1839 ('lressa'), an EGFR
`proliferation and progesterone receptor (PR) expression in normal breast epithe(cid:173)
`lium. Premenopausal normal breast tissue from 8 women was implanted subcu(cid:173)
`taneously into athymic nude mice ·in separate experiments (8 xenografts per
`mouse; median number of mice used per experimer:it = 14). Following 14 days,
`each mouse had two xenografts removed and a 2 mg oestradiol (E2) pellet
`inserted subcutaneously, following whk:h the mice were treated daily with
`ZD1839 (10-100 mg/kg) or vehicle for 14 days. The remaining xenografts were
`removed after 7 and 14 days of treatment. Proliferation and progesterone recep(cid:173)
`tor (PR) expression were assessed by determining the percentage of positive cells
`in 1000 epithelial cell's counted following immunostaining for Ki67 (proliferative
`marker) and PR, respectively. After 14 days' treatment, there was a rise in median
`proliferation and PR expression in the vehicle + E2 group compared with the
`pre-treatment level (5.4 % vs 2.6 %, and 26.2 % vs 14.7 %, respectively; both
`p<0:001 ). In the ZD1839 + E2 group there was a decrease in epithelial prolifer(cid:173)
`ation and a decrease in PR expression compared with the control vehicle + E2
`group (1.1 % ils 5.4%, and 20.1 % vs 26.2%, respectively, both p<0.001). These
`results suggest that o,estrogen-stimulated proliferation and PR expression do
`
`802
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`Proceedings of the American Association for Cancer Research " Volume 42 o March 2001
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`West-Ward Exhibit 1095
`Yu 2001 - Page 002
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