WO2009151908A1

WO2009151908A1 - Biomarkers for egfr/her/erbb drug efficacy

Info

Publication number: WO2009151908A1
Application number: PCT/US2009/044771
Authority: WO
Inventors: Thomas W. Grunt; Waheed Shabbir
Original assignee: Wyeth LLC
Current assignee: Wyeth LLC
Priority date: 2008-05-25
Filing date: 2009-05-21
Publication date: 2009-12-17
Anticipated expiration: 2010-11-25
Also published as: BRPI0911451A2; CA2722890A1; AU2009257802A1

Abstract

The present invention provides biomarkers useful in the determination of a cell's sensitivity to an ErbB2 blocking compound, methods of use of the biomarkers and kits comprising tools for detecting the biomarkers. The biomarkers comprise components of the ErbB3/PI3K/Akt and their phophorylated forms.

Description

BIOMARKERS FOR EGFR/HER/ERBB DRUG EFFICACY

FIELD

The invention relates generally to biomarkers and their use in assessing or predicting cancer therapy efficacy in a patient.

BACKGROUND

ErbB transmembrane proteins belong to the family of growth factor receptor protein tyrosine kinases (RTK). Four members have been described to the ErbB subfamily: ErbB1 (EGFR), ErbB2 (HER-2), ErbB3 (HER-3), and ErbB4 (HER-4). Upon ligand binding, these receptors (with the exception of ErbB2) dimerize and undergo tyrosine phosphorylation, which initiates a signaling cascade leading to critical cellular functions such as proliferation, survival, differentiation and mobility. In the case of ErbB2, it does not have a ligand per se, but rather acts as a heterodimer binding partner and co-receptor for the other ErbB family members. In the case of ErbB3, it has no endogenous tyrosine kinase activity of its own. When ErbB3 binds to its cognate ligand, heregulin, a ErbB2/ErbB3 kinase-active heterodimer is formed, resulting in the activation of the PI3K/Akt cascade, which enables cell proliferation.

Deregulation and hyperactivation of signaling through the EGF family of receptors is associated with many types of cancers. Deregulation of ErbB1 is associated with head and neck, breast, lung, bladder, prostate, brain, pancreatic, ovary, colon and kidney cancers. Deregulation of Erb2 is associated with breast, ovary, lung, prostate, gastric and oral cancers.

Drugs that target the ErbBs in the treatment of cancer are currently in development, in the clinic and on the market. These drugs include biologies, generally monoclonal antibodies, as well as small molecule tyrosine kinase inhibitors (TKIs). ErbB1-directed therapeutic antibodies include e.g. cetuximab (Erbitux®, Imclone, Branchburg, NJ), ABX-EGF (Abgenix), MDX-447 (Mederex) and EMD 72000 (Merck KgA). ErbB1 TKIs, which show some level of inhibiting ErbB2, include e.g. gefitinib, erlotinib and pilitinib (a.k.a. EKB-569, see Torrance et al., Nature Medicine, 6(8): 1024-1028, 2000, and U.S. Pat. Nos. 6,002,008 and 6,297,258, which are incorporated herein by reference.) ErbB2-directed therapeutic antibodies include e.g. trastuzumab (Herceptin®, Genentech) and pertuzumab (Omnitarg™, Genentech). ErbB2 TKIs include e.g. CP-654,577 (European Pat. No. EP1029853) and CP-724,714 (£-2-Methoxy-Λ/-(3-{4-[3-methyl-4-(6-methyl-pyridin-3-yloxy)- phenylamino]-quinazolin-6-yl}-allyl)-acetamide). Tyrosine kinase inhibitors of multiple ErbBs include e.g. canertinib (CI-1033; a 4-anilinoquinazoline acrylamide derivative), HKI-272 (a 6,7-disubstituted-4-anilinoquinoline-3-carbonitrile), lapatinib (GW572016, PCT Pub. No. WO9935146), PKI-166 (R)-4-[4-[(1-phenylethyl)amine]-7H-pyrrolo[2,3- d]pyrimidin-6-yl]-phenol), and AEE788 (Traxler et al., Cancer Research, 64:4931- 4941 , 2004.) For a review of ErbB-family inhibitors, see Rabindran, Cancer Letters, 227:9-23, 2005, which is incorporated herein by reference.

As mentioned above, ErbB1 and 2 are overexpressed or hyperactivated in many tumors, including ovarian and breast cancer. They stimulate carcinogenesis and malignant progression, and confer unfavorable prognoses. Clinical success has recently been obtained by targeting ErbB2 in ErbB2+ breast cancers. However, only approximately 30% of ErbB2+ breast cancers respond to targeted ErbB2 blockade and most of the responders eventually develop secondary resistance.

SUMMARY

The invention provides molecular markers (biomarkers) that enable the determination or prediction of whether a particular cancer having an ErbB phenotype can respond favorably to a particular ErbB modulating drug. The inventors have made the surprising discovery that biomarkers in the ErbB3 pathway are useful in determining the sensitivity of cells to ErbB1/ErbB2 blockers and kinase inhibitors. In one aspect, the invention provides a method of determining the sensitivity of a cell to a compound that modulates ErbB2 activity, comprising the steps of (a) contacting the cell with the compound; (b) determining the phosphorylation status of one or more components of an ErbB3 signaling pathway; and (c) comparing the phosphorylation status of the one or more components of the ErbB3 signaling pathway obtained in step (b) to a reference phosphorylation status of the one or more components of the ErbB3 signaling pathway, wherein a difference between the phosphorylation status of the one or more component of an ErbB3 signaling pathway obtained in step (b) and the reference phosphorylation status of the one or more components of the ErbB3 signaling pathway indicates that the cell is sensitive to the compound. In some embodiments, the result of the comparison of step (c) is provided to a user in a readable format, such as a written report or digital format. In some embodiments, the compound inhibits ErbB2 kinase activity. In one embodiment, the compound is (2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7- ethoxyquinolin-6-yl}-4-(dimethylamino)but-2-enamide (EKB-569, a.k.a. pilitinib.)

In some embodiments, the cell is a cancer cell, such as a breast cancer cell or an ovary cancer cell. In some embodiments the cancer cell is from a cell line, such as e.g. BT-20, BT-474, MCF-7, MDA-MB-231 , MDA-MB-361 , MDA-MB-453, MDA-MB- 468, SKBR-3, SW-527, T-47D, ZR-75-30, A-2774, A-2780, A-2780-ADR, CAOV3, H- 134, HEY, HOC-7, OVCAR3, PA1 , SKOV3, and TR-170. In other embodiments, the cell is obtained from a tumor in a patient. In some embodiments, the components of the ErbB3 pathway include ErbB3, protein kinase B (Akt), phosphatidylinositol-3-OH kinase (PI3K), glycogen synthase 3 (GSK3), and phosphoinositide phosphatase (PTEN).

In one embodiment, the phosphorylation status of ErbB3 is determined by detecting the relative level of pTyr1289-ErbB3 in the cell. A reduction in relative ErbB3 phosphorylation indicates that the cell is predicted to be sensitive to the antiproliferative effects of the ErbB2 kinase inhibitor. In another embodiment, the phosphorylation status of Akt is determined by detecting the relative level of pSer473pThr308-Akt in the cell. A reduction in relative Akt phosphorylation indicates that the cell is predicted to be sensitive to the antiproliferative effects of the ErbB2 kinase inhibitor. In another embodiment, the phosphorylation status of GSK3β is determined by detecting the relative level of pSer9-GSK3β in the cell. A reduction in relative GSK3β phosphorylation indicates that the cell is predicted to be sensitive to the antiproliferative effects of the ErbB2 kinase inhibitor. In yet another embodiment, the phosphorylation status of PTEN is determined by detecting the relative level of pSer380-PTEN in the cell. An increase in relative PTEN phosphorylation indicates that the cell is predicted to be sensitive to the antiproliferative effects of the ErbB2 kinase inhibitor. In some embodiments, the reference phosphorylation status of one or more components of the ErbB3 pathway is established in cells treated with a ErbB2 modulating compound, wherein the cells are resistant to the ErbB2 modulating compound. For example (which is generalizable to any and all biomarkers of the invention), in the embodiment of using GSK3 as the biomarker, (a) the level of pSer9- GSK3β is determined in the test cell after contact with the compound, (b) the level of pSer9-GSK3β is determined in a compound-resistant cell after contact with the compound, and (c) the levels of pSer9-GSK3β determined in each cell are compared. In some embodiments, the levels of the phosphorylated ErbB3 pathway components are standardized to a standard. In some embodiments, the standard can be a constitutive protein such as e.g. actin or GAPDH. In other embodiments, the standard can be the level of unphosphorylated ErbB3 pathway component, or the total level of phosphorylated and unphosphorylated ErbB3 pathway component.

In another aspect, the invention provides for the use of an ErbB3 pathway phosphorylation assay to determine whether a tumor can respond to the antiproliferative effects of an ErbB2 blocking compound. In some embodiments, the phosphorylation assay determines the phosphorylation status of members of the

ErbB3 pathway in a cell obtained from a tumor and after the cell was treated with an

ErbB2 blocking compound, which in turn correlates to the tumor's sensitivity to the ErbB2 blocking compound.

In another aspect, the invention provides a kit that is useful in assessing the sensitivity of a cell to an ErbB2 blocking agent. The kits includes (a) at least one antibody that specifically binds to the phosphorylated form ErbB3.

DRAWINGS

Figure 1 depicts a line graph showing the proliferation response of breast cancer cell lines to increasing doses of pelitinib.

Figure 2 depicts a line graph showing the proliferation response of ovarian cancer cell lines to increasing doses of pelitinib. Figure 3 depicts Western blots indicating the levels of EGFR and actin in breast cancer cell lines (upper panel) and ovarian cancer cell lines (lower two panels.)

Figure 4 depicts Western blots indicating the levels of ErbB2 and actin in breast cancer cell lines (upper panel) and ovarian cancer cell lines (lower two panels.) Figure 5 depicts the correlation between relative EGFR and ErbB2 protein expression level and antiproliverative efficacy of pelitinib in breast (upper panels) and ovarian (lower panels) cancer cell lines.

Figure 6 depicts the dose-response relationship of in vitro growth inhibition of

SKBR-3 (pelitinib-sensitive - upper left panel) and T47D (pelitinib-resistant - upper right panel) breast cancer cell lines. The lower panels depict Western blots of the time-dependent effects of pelitinib (0.1 μM) in pelitinib-sensitive SKBR3 (left panels) and pelitinib-resistant T47D (right panels) breast cancer cell lines.

Figure 7 (upper panel) depicts a line graph showing the proliferation response of transfected SKBR3 cells, which contain either myr-AKT, an empty vector, or a GFP construct, to increasing doses of pelitinib. The lower panel depicts a Western blot showing phosphor-AKT expression, AKT, and actin expression in the transfected

SKBR3 cells exposed to pelitinib.

DETAILED DESCRIPTION

An object of the invention is the assessment of a tumor cell's ability to respond to targeted ErbB2 blockade. The benefit to being able to predict whether a particular cancer will respond to a given type of therapy is useful for determining the safety and effectiveness of cancer treatment. When a practitioner is able to select the therapy most likely to work against a particular disease, any unnecessary risk and expense associated with misdirected therapy is reduced. In one aspect, the invention provides a method for determining or predicting the ability of a cell to respond to therapy that blocks ErbB2 signaling, by assessing the phosphorylation status of components of the ErbB3 pathway. The cell that is tested can be any cell. In some embodiments, the cell is a tumor cell, such as e.g. an ovarian cancer cell or breast cancer cell. The cell can be from a cell line, a primary cell line or from a biopsy of a tumor from a patient. The therapy can be any compound or set of conditions that blocks the ErbB2 pathway.

In another aspect, the invention provides for the use of a phosphorylation assay for ErbB3 pathway components in the diagnosis or prognosis of a tumor's response to ErbB2 blocking compounds. In some embodiments, a cell is obtained from a tumor in a patient and contacted with an ErbB2 blocking compound. The phosphorylation status of one or more ErbB3 pathway components in the cell is determined. Generally, in the case in which an activating component of the ErbB3 pathway shows reduced phosphorylation, relative to a reference, then the diagnosis or prognosis of the tumor's response to ErbB2 blocking compounds is positive, i.e., the tumor is sensitive to the compound. For example, a reduced level of pSer9- GSK3β indicates that the tumor is sensitive to the compound. Generally, in the case in which inactivating components of the ErbB3 pathway show increased phosphorylation, relative to a reference, then the diagnosis or prognosis of the tumor's response to ErbB2 blocking compounds is positive, i.e., the tumor is sensitive to the compound. For example, an increased level of pSer380-PTEN indicates that the tumor is sensitive to the compound.

In yet another aspect, the invention provides a kit useful in the determination of a cell's sensitivity to an ErbB2 blocking agent. The kit includes an agent that ascertains the phosphorylation status of an ErbB3 pathway component and instructions for determining whether the test cell is sensitive to an ErbB2 blocking compound. In one embodiment, the kit includes an antibody specific to a phosphorylated form of an ErbB3 pathway component.

As used herein, "component of an ErbB3 pathway" means any one or more of an upstream ligand of ErbB3, binding partner of ErbB3, and/or downstream effector molecule that is modulated through ErbB3. Non-limiting examples of ErbB3 pathway components include heregulin, ErbB3/HER3, phosphatidylinositol-3-OH kinase

(PI3K), protein kinase B (Akt or PKB), HER3 phosphatase, human epidermal growth factor receptor tyrosine kinase inhibitor (HER TKI), HER2, phosphatase and tensin homolog (PTEN), phosphatidylinositol 4,5-bisphosphate (PIP₂), phosphatidylinositol

3,4,5-trisphosphate (PIP3), p27, and phosphoinositide-dependent kinase 1 and 2

(PDK1 and PDK2). "Activating components" of the ErbB3 pathway are those components, which when phosphorylated, stimulate cell proliferation and/or, protein sysnthesis. Examples include PI3K, GSK3 and Akt. "Inactivating components" of the ErbB3 pathway are those components, which when phosphorylated, inhibit cell proliferation and/or, protein synthesis. An example of an inactivating component is PTEN. See also Planchon et al., Journal of Cell science, 121 (3):249-253, 2008; Menedez and Lupu, Breast Cancer Research, 9:1 11-115, 2007; and Osaki et al., Apoptosis, 9:667-676, 2004, which are incorporated in their entirety herein by reference.

As used herein, "phosphorylation status" refers to whether a molecular entity is phosphorylated, and/or to what extent the molecular entity is phosphorylated. As the skilled artisan readily appreciates, phosphorylation and dephosphorylation of proteins, lipids and carbohydrates can determine the activity of the protein, lipid or carbohydrate. Kinases catalyze the addition of a phosphate group to a protein, such as on a tyrosine, serine and/or threonine residue, or on a lipid, such as a phosphoinositol, whereas phosphatases catalyze the removal of phosphate groups.

As "phosphorylation status" pertains to the present invention, it is generally known in the art that the ErbB proteins are tyrosine kinases that activate kinase/phosphatase signaling cascades. In the case of ErbB3, (a) activated ErbB3 is phosphorylated at least at tyrosine 1289, which (b) phosphorylates and activates PI3K, which (c) mediates the phosphorylation of PIP2 to PIP3, which (d) recruits PKB/Akt and PDK1 to the plasma membrane. PKB/Akt is activated by being phosphorylated at serine 473 by PDK2 and at threonine 308 by PDK1. Activated PKB/Akt phosphorylates the constitutively active GSK3 at least at serine 9, thereby inactivating it. Active non-phosphorylated GSK3 degrades mitogenic β-catenin and inhibits protein synthesis, hence, phosphorylated (inactivated) GSK3 allows for transcription and protein sysnthesis to proceed. PTEN, which is a phosphoinositide phosphatase, See also Farrar et al., Aging Cell, 4:1-12, 2005, which is incorporated herein by reference.

Phosphorylation status can be determined in myriad ways. For example, it is well known in the art that phosphorylated proteins can be detected via immunoassays using antibodies that specifically recognize the phosphorylated form of the protein

(see, e.g., Lin et al., Br. J. Cancer, 93:1372-1381 , 2005, which is incorporated herein by reference). Immunoassays generally include immunoblotting (e.g., Western blotting), RIAs and ELISAs. More specific types of immunoassays include antigen capture/antigen competition, antibody capture/antigen competition, two-antibody sandwiches, antibody capture/antibody excess, and antibody capture/antigen excess. Immunoassays and methods of making antibodies are described in Harlow and Lane, Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, which is incorporated herein by reference. Phospho- specifc antibodies can be made de novo or obtained from commercial or noncommercial sources. For example, anti-phospho-GSK-3β (Ser 9) can be obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Examples of the effective use of that antibody are depicted in Endo et al., Stroke, 37: 2140-2146, 2006; Capozza et al., Am. J. Physiol. Cell Physiol. 288: C1317-C1331 , 2005; Rauch et al., Am. J. Physiol. Cell Physiol. 288: C593-C605, 2005; and Maier et al., FASEB J., 19: 1353-1355, 2005, which are incorporated herein by reference. Phosphorylation status can also be determined by metabolically labeling cells with radioactive phosphate in the form of [γ-³²P]ATP or [γ-³³P]ATP. Phosphorylated proteins or lipids become radioactive and hence traceable and quantifiable through scintillation counting, radiography, and the like (see Wang and Koshland, J. Biol. Chem., 253:7605-7608, 1978, which is incorporated herein by reference.) For example, metabolically labeled proteins can be extracted from cells, separated by gel electrophoresis, transferred to a membrane, probed with an antibody specific for a particular ErbB3 pathway component and subjected to autoradiography to detect ³²P or ³³P. Alternatively, the gel can be subjected to autoradiography prior to membrane transference and antibody probing. As used herein, "compound that modulates ErbB2 activity" means a compound that either activates the ErbB2 pathway or inhibits the ErbB2 pathway. As used herein, ΕrbB2 blocking compound" or "compound that blocks the ErbB2 pathway" means a compound that blocks signaling through ErbB2. Examples include antibodies such as trastuzumab and pertuzumab, and small molecule RTK-inhibitors such as gefitinib, erlotinib, pilitinib, CP-654,577, CP-724,714, canertinib, HKI-272, lapatinib, PKI-166, and AEE788. EXAMPLE 1 : CELL PROLIFERATION

The effects of the irreversible ErbB inhibitor pelitinib on the growth activity and on ErbB-triggered signaling in 1 1 human breast and 1 1 human ovarian cancer cell lines was examined using the MTT assay (see Mosmann, J. Immunol. Meth. 65:55-63, 1983 and Wilson, A. P., Cytotoxicity and Viability Assays in Animal Cell Culture: A Practical Approach, 3rd ed. (ed. Masters, J. R. W.) Oxford University Press: Oxford 2000, Vol. 1 , which are incorporated herein by reference) and Western blotting, respectively. Table 1 lists the ICso-values for in vitro growth inhibition by pelitinib. Those IC₅₀-values vary over a wide range among the individual breast and ovarian cancer cell lines tested. The data presented in Table 1 were obtained from MTT assays after 72 h of drug exposure. The means of 3 separate experiments are provided, the standard deviations of which were always less than 25% of the mean.

TABLE 1 : Pelitinib IC50 Antiproliferation Effects

SKBR3 and T47D were identified as most sensitive and most resistant breast cancer cell lines, respectively. In contrast, the sensitivity of the ovarian cancer cell lines was more evenly distributed (Tables 2 & 3; Figs. 1 & 2).

As shown in Fig. 1 and Table 2, the small ErbB tyrosine kinase inhibitory drug pelitinib (EKB-569) inhibited the in vitro growth of breast cancer cell lines in a dose- dependent manner, but the individual cell lines revealed distinct sensitivities against the inhibitor. The cell numbers were determined by MTT assay after 72 hours of drug exposure. Results are means of three independent experiments. SD was always less than 30% of the mean.

As shown in Fig. 2 and Table 3, pelitinib also inhibited the in vitro growth of ovarian cancer cell lines in a dose-dependent manner, but the individual cell lines revealed distinct sensitivities against the inhibitor. The cell numbers were determined by MTT assay after 72 hours of drug exposure. Results are means of three independent experiments. SD was always less than 30% of the mean.

TABLE 2: Breast Cancer Cell Line Cell Numbers as Percentage of Control

TABLE 3: Ovarian Cancer Cell Line Cell Numbers as Percentage of Control

EXAMPLE 2: ErbB SIGNALING

Interestingly, the antiproliferative activity of the drug did not correlate with EGFR and ErbB2 protein levels, as shown in Figures 3-5. Figure 3 depicts the baseline levels of EGFR protein expression in breast (upper panel) and ovarian cancer cell lines (lower panels) as demonstrated by Western blot analysis using enhanced chemiluminescence. Figure 4 depicts the baseline levels of ErbB2 protein expression in breast (upper panel) and ovarian cancer cell lines (lower panels) as demonstrated by Western blot analysis using enhanced chemiluminescence. In both experiments, BT-474 breast cancer cells were used as reference EGFR-positive and ErbB2-positive cells, respectively, in all Western analyses. Membranes were semi- quantitatively evaluated by densitometry. EGFR and ErbB2 bands were related to actin bands. In BT-474 cells, the resulting EGFR/actin and ErbB2/actin ratios were arbitrarily set at 1.0 and the ratios of all other cell lines were related to it and are shown below each autoradiograph.

Figure 5 depicts the correlation between relative EGFR and ErbB2 protein expression level (semi-quantitative) and antiproliferative efficacy of pelitinib (IC50 values) in breast (upper panels) and ovarian (lower panels) cancer cell lines. Each dot in the graphs represents a single cell line. On the X-axis are the EGFR or ErbB2 levels (relative to BT-474 = 100) and on the Y-axis the IC50 values of each cell line are given.

Moreover, drug-dependent inhibition of EGFR, of ErbB2 and of ERK1/2 phosphorylation was seen in both pelitinib-sensitive and pelitinib-resistant cells indicating that inhibition of ERK1/2 downstream signaling is not sufficient for drug- dependent growth arrest. In contrast, phosphorylation of ErbB3 at Tyr1289, of AKT at Ser473 and at Thr308, and of GSK3beta at Ser9 was blocked only in the sensitive, but not in the resistant cells (Fig. 6). Conversely, pelitinib rapidly induced phosphorylation of PTEN at Ser380 in sensitive, but not in resistant cells (Fig. 6). The upper panel of Figure 6 provides a dose-response relationship of in vitro growth inhibition of SKBR-3 (pelitinib-sensitive - left panel) and T47D (pelitinib- resistant - right panel) breast cancer cell lines. In that experiment, cell numbers were determined by the MTT assay after 72 hours of drug exposure. The results provided are the means of three independent experiments, in which the standard deviations were always less than 30% of the mean. The lower panels provide a time- dependent effect of the ErbB tyrosine kinase inhibitor pelitinib (0.1 μM) on the expression and activity (phosphorylation) of the ErbB receptors and of the ErbB downstream signaling mediators PTEN, AKT, GSK-3β, and ERK in pelitinib-sensitive SKBR3 (left panels) and pelitinib-resistant T47D (right panels) breast cancer cell lines. In both cell lines, pelitinib stably repressed the phosphorylation of EGFR (pEGFR), whereas it downregulated pErbB2 only moderately. A strong and transient reduction of pErbB3 was only seen in the pelitinib-sensitve SKBR3, but not in the pelitinib-resistant T47D cells. Moreover, phosphorylation of AKT at Ser473 and Thr308 and of GSK3beta at Ser9 was strongly inhibited in pelitinib-sensitive, but not in pelitinib-resistant cells. In contrast, pelitinib rapidly induced the phosphorylation of PTEN at Ser380 in sensitive, but not in resistant cells, whereas pERK1/2 levels were reduced in both pelitinib-sensitive and pelitinib-resistant cells. Actin was used as a loading control.

Furthermore, ectopic expression of constitutively active AKT in the normally pelitinib-sensitive SKBR-3 cells conferred a pelitinib-resistance phenotype to the transfected SKBR-3 cells (Fig. 7). Figure 7 provides the data showing that the transfection of constitutively active

AKT confered resistance against the ErbB tyrosine kinase inhibitor pelitinib on SKBR3 breast cancer cells. In this experiment, pelitinib sensitive SKBR3 cells were transfected with pCMV6 containing no insert (empty vector), myristoylated AKT (myr- AKT), or green fluorescent protein (GFP). MTT assays revealed that myr-AKT transfected SKBR3 cells continued to grow in the presence of increasing concentrations of pelitinib, whereas growth of vector and GFP transfected cells remained inhibited by pelitinib (upper panel). Western blot analysis revealed the increased AKT protein expression and phosphorylation at Ser473 and Thr308 in myr- AKT transfected cells, but not in vector or GFP transfected cells (lower panel). Again, actin was used as a loading control.

Taken together, the data demonstrated that ErbB3/PI3K/AKT, but not ERK1/2 signaling plays a crucial role in determining sensitivity/resistance of the cells against the irreversible dual EGFR/ErbB2 inhibitor pelitinib. Therefore, we propose that drug- mediated downregulation of phospho-AKT and phospho-ErbB3 levels as surrogate markers for ErbB drug efficacy in breast cancer. EXAMPLE 3: METHODS - CELL LINES

The following human breast carcinoma cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA): BT20, BT474, MCF7, MDA-MB-231 , MDA-MB-361 , MDA-MB-453, MDA-MB-468, SKBR3, SW527, T47D, ZR-75-30.

The human ovarian carcinoma cell lines CAOV3 and H 134, as well as the ovarian teratocarcinoma cell line PA1 were also obtained from the ATCC. Additional human ovarian carcinoma cell lines used were: OVCAR3 and SKOV3, from the National Institutes of Health; A2774 from C. Marth, Med. Univ. Innsbruck, Austria; A2780 and A2780ADR (Adriamycin resistant) from M. Krainer, Med. Univ. Vienna, Austria; HEY and HOC7 from R. Buick, Univ. Toronto, Canada; and TR170 from B. Hill, Imperial Cancer Research Fund, London, UK.

All cell culture work was performed under sterile conditions (sterile guard hood Gelarie Class 100 [1-20090-OPERA]). The cell lines were cultured at 37°C, with 5% CO₂ and 95% relative humidity using a CO₂ water jacketed incubator for mammalian cell culture (Cytoperm2) from Heraeus Instruments (Duesseldorf, Germany). The culture of all incubated human cells was controlled every day to observe confluence status by using a light microscope (Fluovert, Leitz, Wetzlar, Germany). The culture medium with 10% (v/v) FCS, 100 IU (μg)/ml penicillin-streptomycin, and 2 mM glutamine (Gibco, Karlsruhe, Germany) was replaced every third day of culture. Cultured cells were used shortly after plating and expansion to avoid long-term effects, such as mutations.

Splitting of cell lines was performed by washing the monolayers with PBS solution and incubating them with 1 - 3ml trypsin/EDTA for 1 - 5 min depending on the size of the culture. After cells had been detached, medium with 10% FCS was added and cells were gently resuspended and split to a ratio 1 : 3 or 1 : 5 depending on the growth rate of the cell line.

Cell lines were stored in liquid nitrogen. Cells were trypsinized when confluence reached 70%, centrifuged at IOOOrpm for 5 min, resuspended in ice cold freezing solution (growth medium containing 15% FCS and 10% (v/v) DMSO) and aliquoted in cryotubes, which were put into a styrofoam box and transferred into a -8O⁰C freezer for gradual cooling. The following day, the frozen tubes were transferred into liquid nitrogen for long-term storage.

Frozen cell lines were thawed at room temperature in hand-warm water.

Immediately after thawing, the cell solution was transferred into a 50 ml centrifuge tube and resuspended in medium with 10% FCS and centrifuged at IOOOrpm for

5min. The supernatant was then discarded and cell pellet was resuspended in fresh medium with 10% FCS and plated.

EXAMPLE 4: METHODS - CELL PROLIFERATION ASSAYS

1500 cells/well were plated in 96-well plates in medium containing 10% FCS. After overnight incubation, drug was added in serum-free medium to get the final concentration of 5% FCS. The cells were exposed for 72 hours to pelitinib (Pelitinib (EKB-569) was obtained from Wyeth, Collegeville, PA, USA.)

The proliferation rate of all cell lines was assayed by the EZ4U Nonradioactive Cell Proliferation Assay (a.k.a. MTT assay; Biomedica, Vienna, Austria). This assay depends on the reduction of non-toxic tetrazolium salt into intensely coloured formazan derivatives. This reduction requires functional mitochondria, which are inactivated within a few minutes after cell death. In this assay, cultured cells were incubated for the last 3 - 4 h with EZ4U, and formazan production, which is linearly related to cell number, was assayed by measuring absorbance at 490/620 nm wavelength in a microplate photometer. Calibration curves showed that a highly significant correlation occurred between optical density and the number of cells.

Correlation analyses between EGFR or ErbB2 and drug-mediated growth inhibition (IC₅O values), respectively, were performed to examine a possible relationship between these variables. All three variables were measured in different experiments. The coefficient of determination (or Pearson r) indicates the strength and direction of magnitude of correlation. A value of one (1 ) means increasing negative values or r indicates negative correlation between the variables, r² indicates the quality of coefficient of determination. Usually this value ranges from 0 to 1 ; e.g. r² = 0.593 indicates that 59% of variance in X can be explained by variance in Y, likewise 59% of variance in Y can be explained by variance in X. 95% confidence interval indicated that all values lie within this range. Two-tailed p-values were determined. The smaller the p-value, the more significant the relationship; e.g. if the p-value is 0.001 , the correlation is highly significant (symbol: ^***), if the p-value is 0.01 , the correlation is moderately significant (symbol: ^**), and if the p-value is 0.05, the correlation is of low significance (symbol: ^*). The p-value summary gives the answer to the question whether the correlation is significant. The analysis was done using GRAPHPAD™ PRISM software (GraphPad Software, Inc., La JoIIa, CA.)

EXAMPLE 5: METHODS - PROTEIN EXPRESSION AND I M M U N OBLOTTI N G

3x10⁵ cells were plated in 60 mm dishes in DMEM containing 10% FCS, 100 IU (μg)/ml penicillin-streptomycin, and 2 mM glutamine. After overnight incubation, drugs were added in serum-free medium in order to obtain a final concentration of 5% FCS. After treatment, cells were washed twice with ice-cold PBS. Cold modified RIPA buffer (15OmM NaCI, 5OmM Tris pH 7.4, 0.5% Na-deoxycholate, 2mM EGTA, 5mM EDTA, pH 7.4, 3OmM NaF, 4OmM β-Glycerophosphate, pH7.2, 1OmM tetrasodium pyrophosphate, 3mM Benzamidine, 1% Nonidet P-40, 2 mM Na- Orthovanadate) was applied. After 5 minutes incubation on ice, the cells were scraped using a cell scraper and transferred into 1.5 - 2.0 ml Eppendorf tubes, vortexed several times and left on ice for another 5 minutes. The cell lysate was centrifuged at 12,500 rpm in a microfuge at 4°C for 30 minutes. The supernatant was transferred into new Eppendorf tubes and stored at -80⁰C. After having determined the protein concentrations of each sample using the Bradford method (Bradford, Anal. Biochem. 72:248-254, 1976), 4 x sample buffer (50% Glycerol, 125mM Tris- HCI, pH 6.8, 4% SDS, 0.125% Bromophenol blue, 5% Beta-mercaptoethanol) was added to the lysate and suitable amounts of protein were loaded onto an SDS- polyacrylamide gel for electrophoresis. For construction of a standard curve BSA solutions with concentrations between 0.156 and 10 μg/μl were used. Each standard and sample was measured 3 times and the average values were calculated. The final concentration of the protein samples was adjusted to 1 μg/μl with 3 volumes modified RIPA and 1 volume of 4x sample buffer. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins by molecular weight. The MINI PROTEAN II™ equipment from Bio-Rad, Inc. was used for polyacryamide gel electrophoresis. 20 μg of samples (1 μg/μl) were loaded in each slot. 1 μl of marker (MAGIC MARK XP, Invitrogen) was used in the first slot. After electrophoesis, proteins were transferred to a polyvinylidenedifluoride (PVDF) membrane using a protein wet transfer chamber from Bio-Rad (Trans-blot-cell wet transfer apparatus) in 1X transfer buffer (15OmM Glycine, 5OmM Tris pH8.3, 0.05% SDS, 20% Methanol.)

The membrane was transferred to a small container with blocking solution (4% BSA, 5OmM Tris pH7.5, 15OmM NaCI, 0.1 % Tween20) and incubated for 1 hour at room temperature on a shaker to decrease unspecific binding of the primary antibody. The next step included three washings in TBS-T (5OmM Tris pH7.5, 15OmM NaCI, 0.1 % Tween20) for 15 minutes. Immediately after blocking and washing, the diluted primary antibody was added and incubated in a closed plastic container at room temperature for 2 hours. After that, 2 x 5 minutes and 2 x 10 minutes washing with TBS-T was done followed by incubation in the secondary antibody for 1 hour at room temperature. The membrane was then washed again 2 x 5 minutes and 2 x 10 minutes in TBS-T, and then 2 x 5 min and 2 x 10 minutes in TBS. As a rule, phosphospecific antibodies were always used in the first round of immunoblotting. For development of the membrane the WESTERN-BLOT ECL DETECTION KIT was used according to the manufacturer (Amersham Biosciences). After 5 minutes, the blot was drained on paper towel, warped in saran wrap, and exposed in an X-ray cartridge to a sheet of autoradiography film until optimum signals were obtained. The autoradiographs were scanned and processed using ADOBE PHOTOSHOP 6.0 software (Adobe).

Antibodies used to detect the phosphorylated proteins were provided in Phospho- Erk1/2 Pathway Sampler Kit and the Phospho-Akt Pathway Sampler Kit (Cell

Signaling, MA, USA). The antibodies were diluted in TBS-T plus 1 % bovine serum albumin (BSA). Primary antibody solutions also contained 0.05% Na-azide. Table 4 provides a list of the antibodies used in this study: TABLE 4

MW(kDa) of Dilution Source the protein species

Phospho-c-Raf (Ser338) (56A6) rabbit monoclonal 74 1:1000 Rabbit antibody

Phospho-MEK1/2 (Ser217/221) antibody 45 1:1000 Rabbit

Phospho-p44/42 MAP kinase (Thr202/Tyr204) 42,44 1:1000 Rabbit antibody

Phospho-p90RSK (Ser380) antibody 90 1:1000 Rabbit

Phospho-Elk-1 (Ser383) antibody 62 1:1000 Rabbit

Anti-rabbit IgG, HRP-linked antibody 1:1000 Goat

Phospho-Akt (Ser473) antibody 60 1:1000 Rabbit

Akt antibody 60 1:1000 Rabbit

Phospho-Akt (Thr308) antibody 60 1:1000 Rabbit

Phospho-c-Raf (Ser259) antibody 74 1:1000 Rabbit

Phospho-GSK-3β (Ser9) antibody 46 1:1000 Rabbit

Phospho-PTEN (Ser380) antibody 54 1:1000 Rabbit

Phospho-PDK1 (Ser241) antibody 58-68 1:1000 Rabbit

Anti-rabbit IgG, HRP-linked antibody 1:1000 Goat

Erk1/2 antibody (Upstate) 44/42 1:1000 Rabbit

EGFR antibody (Santa Cruz) 170 Rabbit pEGFR antibody (Santa Cruz) 170 1:1000 Goat

ErbB2 antibody (Santa Cruz) 185 1:1000 Rabbit pErbB2 antibody (Santa Cruz) 185 1:500 Rabbit

ErbB3 antibody (Santa Cruz) 185 1:500 Rabbit pErbB3 antibody (Cell Signaling) 185 1:1000 Rabbit

Actin antibody (Santa Cruz) 40 1:500 Goat

Anti-rabbit IgG, HRP-linked antibody (Promega ) 1:1500 Donkey

Anti-goat IgG, HRP-linked antibody (Promega) 1:1500 Donkey

Claims

WHAT IS CLAIMED IS:

1. A method of determining the sensitivity of a cell to a compound that modulates ErbB2 activity, comprising:

(a) contacting the cell with the compound;

(b) determining the phosphorylation status of one or more components of an

ErbB3 signaling pathway; and

(c) comparing the phosphorylation status of the one or more components of the ErbB3 signaling pathway obtained in step (b) to a reference phosphorylation status of the one or more components of the ErbB3 signaling pathway;

wherein a difference between the phosphorylation status of the one or more component of an ErbB3 signaling pathway obtained in step (b) and the reference phosphorylation status of the one or more components of the ErbB3 signaling pathway indicates that the cell is sensitive to the compound.

2. The method of claim 1 , wherein the compound inhibits ErbB2 activity.

3. The method of any one of claims 1 and 2, wherein the compound is (2E)-N-{4-[(3- chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxyquinolin-6-yl}-4-(dimethylamino)but-2- enamide ("pelitinib").

4. The method of any one of claims 1-3, wherein the one or more components of an ErbB3 signaling pathway comprises a component selected from the group consisting of ErbB3, phosphatidylinositol 3-kinase ("PI3K"), protein kinase B ("AKT"), glycogen synthase kinase-3 beta ("GSK-3β") and phosphatase and tensin homolog deleted on chromosome 10 ("PTEN").

5. The method of any one of claims 1-4, wherein the one or more components of an ErbB3 signaling pathway comprises PTEN, and the phosphorylation status of the PTEN is an increase in the phosphorylation of serine 380.

6. The method of any one of claims 1-5, wherein the component comprises ErbB3, and the phosphorylation status of the Erb3 is a reduction of phosphorylation of tyrosine 1289.

7. The method of any one of claims 1-6, wherein the component comprises AKT, and the phosphorylation status of the AKT is a reduction of phosphorylation of serine 473 and threonine 308.

8. The method of any one of claims 1-7, wherein the component comprises GSK-3β, and the phosphorylation status of the GSK-3β is a reduction of phosphorylation of serine 9.

9. The method of any one of claims 1-8, wherein the cell is a tumor cell.

10. The method of any one of claims 1-9, wherein the cell is a breast cancer cell.

11. The method of any one of claims 1-9, wherein the cell is an ovarian cancer cell.

12. The method of any one of claims 1-1 1 , wherein the cell is obtained from a patient.

13. The method of claim 10, wherein the cell is selected from the group consisting of BT-20, BT-474, MCF-7, MDA-MB-231 , MDA-MB-361 , MDA-MB-453, MDA-MB-468, SKBR-3, SW-527, T-47D, and ZR-75-30.

14. The method of claim 1 1 , wherein the cell is selected from the group consisting of A-2774, A-2780, A-2780-ADR, CAOV3, H-134, HEY, HOC-7, OVCAR3, PA1 , SKOV3, and TR-170.

15. The method of any one of claims 1-14, wherein the reference phosphorylation status of the one or more components of an ErbB3 signaling pathway is obtained from a pelitinib-resistant cell treated with pelitinib.

16. The method of claim 15, wherein the pelitinib-resistant cell is T-47D.

17. The method of any one of claims 1-14, wherein the reference phosphorylation status of the one or more components of an ErbB3 signaling pathway is obtained from a cell having an unactivated ErbB3.

18. The method of any one of claims 1-14, wherein the reference phosphorylation status of the one or more components of an ErbB3 signaling pathway is obtained from a cell not treated with pelitinib or another EGFR/ErbB2 blocking compound.

19. The use of an ErbB3 phosphorylation assay to determine the sensitivity of a tumor to a ErbB2 blocking compound, wherein the Erb3 phosphorylation assay comprises determining the phosphorylation status of an ErbB3 pathway component in the tumor cell after contact with the ErbB2 blocking compound.

20. The use of an ErbB3 phosphorylation assay according to claim 19, wherein the ErbB3 phosphorylation assay comprises detecting a phosphorylated form of the ErbB3 pathway component.

21. The use of an ErbB3 phosphorylation assay according to claim 20, wherein the phosphorylated form of the ErbB3 pathway component is detected with an antibody specific for the phosphorylated form of the ErbB3 pathway component.

22. The use of an ErbB3 phosphorylation assay according to any one of claims 19- 21 , wherein the ErbB2 blocking compound is selected from the group consisting of trastuzumab, pertuzumab, gefitinib, erlotinib, pilitinib, canertinib, and lapatinib.

23. The use of an ErbB3 phosphorylation assay according to any one of claims 19-

22, wherein the ErbB2 blocking compound is pilitinib.

24. The use of an ErbB3 phosphorylation assay according to any one of claims 19-

23, wherein the ErbB3 pathway component is selected from the group consisting of ErbB3, phosphatidylinositol 3-kinase ("PI3K"), protein kinase B ("AKT"), glycogen synthase kinase-3 beta ("GSK-3β") and phosphatase and tensin homolog deleted on chromosome 10 ("PTEN").

25. The use of an ErbB3 phosphorylation assay according to any one of claims 19-

24, wherein phosphorylation assay comprises contacting a cell obtained from the tumor with the ErbB2 blocking compound prior to determining the phosphorylation status of the ErbB3 pathway component.

26. The use of an ErbB3 phosphorylation assay according to any one of claims 19-

25, wherein the tumor is in a patient.

27. The use of an ErbB3 phosphorylation assay according to any one of claims 19- 26, wherein a decrease in the phosphorylation of ErbB3, PI3K, AKT or GSK-3β from the cell after the cell was treated with the ErbB2 blocking compound indicates that the tumor is sensitive to the ErbB2 blocking compound.

28. The use of an ErbB3 phosphorylation assay according to any one of claims 19- 27, wherein an increase in the phosphorylation of PTEN from the cell after the cell was treated with the ErbB2 blocking compound indicates that the tumor is sensitive to the ErbB2 blocking compound.

29. A kit comprising:

(a) an antibody that specifically binds to a phosphorylated form of an ErbB3 pathway component; (b) instructions for determining the sensitivity of a cell to an ErbB2 blocking compound; and

(c) packaging.

30. The method of any one of claims 1-18, further comprising the step (d) of providing the result of the comparison obtained in step (c) to a user in a readable format.