P De

Abstract P6-04-11: Combination of PI3K-AKT-mTOR and MEK-ERK pathway inhibitors overcome acquired resistance to letrozole in ER+ breast cancer models

Background: Approximately 60–70% of breast cancers (BC) express estrogen receptor (ERα) and/or progesterone receptor (PR), the most common malignancy in women in the US and Europe. Many though not all patients with ER+ BC respond to hormonal therapy (e.g. tamoxifen and aromatase-inhibitor) but a large number will eventually develop resistance to long term therapy. Purpose: Development of resistance to endocrine therapy is a clinical issue in ER+ BC. Studies with human cell lines have shown that upregulation of PI3K-AKT-mTOR and MEK-ERK pathways contributes to resistance to endocrine therapy (Miller TW et al JCO 2011; Sanchez CG et al Breast Cancer Res 2011). Using leterozole-resistant ER+ BC model, we investigated the effects of PI3K-AKT-mTOR pathway inhibitor alone and in combination with MEK1/2 inhibitor. Methodology: The anti-proliferative, anti-migratory, and intracellular signaling (pAKT, pS6RP, p4EBP1 and pERK) effects of GDC-0941 (Class I PI3K inhibitor) or GDC-0980 (dual PI3K/mTOR inhibitor) alone and in combination with GDC-0973 (MEK1/2 inhibitor) were evaluated in aromatase overexpressed (MCF-7aro), letrozole-resistant (MCF-7aro/LetR) and long-term-estrogen-deprived (LTEDaro) BC cell lines by MTT assays, integrin-mediated migration assay and Western blots. Results: 1) Both GDC-0980 and GDC-0941 exhibited excellent in vitro cell killing activity in MTT assay in all cell lines with IC509s 10–30 nM & 0.2–1.5 µM respectively. PI3K pathway inhibitors were more potent when combined with GDC-0973; 2) Integrin-mediated migration was significantly higher in both MCF-7aro/LetR andLTEDaro cells than MCF-7 and MCF-7aro cells; 3) Both MCF-7aro/LetR and LTEDaro cells movement were significantly abrogated following the treatment of GDC-0980, and the combination of GDC-0980 plus GDC-0973 more effectively blocked integrin-mediated migration; 4) Inhibition of phosphorylation of AKT (S473/T308), S6RP and 4EBP1 (T37/46) was observed following PI3K pathway inhibitors; 5) pERK was upregulated following the treatment of GDC-0941 (PI3K inhibitor); 6) Basal level of pERK was significantly high in MCF7/LetR and MCF7/LTED cells, and GDC-0980 failed to change the level of pERK in those cell lines; and 7) Combination of PI3K pathway inhibitor plus MEK1/2 inhibitor significantly blocked activation of both pathways. Conclusion: Our in vitro data suggest that therapeutic targeting of the PI3K-AKT-mTOR and the MEK-ERK signaling pathways should be effective in abrogating aromatase-inhibitor-resistance in ER+ BC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-11.

Abstract CT053: High rates of personalized molecular matching are achievable in a precision oncology navigation trial: the I-PREDICT study

Precision medicine has evolved as an individualized approach for treating cancer patients and has become standard in an ever-increasing number of clinical settings. It is predicated upon matching targeted-/immuno-therapy to genomic alterations detected in patients9 tumors. However, widespread feasibility/adoption has been limited by: 1) high rates of insufficient tumor DNA (reaching 25%); 2) panels limited to few genes that are unable to detect multiple classes of genomic alterations; 3) testing patients late in the disease course; and 4) low molecular matching rates, which may be in part due to limited access to trials and the unpredictable nature of genomic alterations detected in each individual. We evaluated the feasibility of investigating molecular profile-related evidence for determining individualized cancer therapy (I-PREDICT) in patients with lethal tumors (NCT02534675). This navigation trial was performed under the auspices of 2 precision medicine programs (UCSD and Avera Cancer Institute) and an IRB-approved protocol. Treatment-naive and previously treated patients with ECOG Citation Format: Jason K. Sicklick, Brian Leyland-Jones, Shumei Kato, Casey Williams, Pradip De, Gregory Heestand, Steven Plaxe, Benjamin Solomon, Vincent Miller, Adam Benson, Jennifer Webster, Jeffrey Ross, Michael Scur, Robert Porter, Shelby Jepperson, Paul Fanta, Razelle Kurzrock. High rates of personalized molecular matching are achievable in a precision oncology navigation trial: the I-PREDICT study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT053. doi:10.1158/1538-7445.AM2017-CT053

Abstract P5-03-14: MLN0128 regulates survival signaling by AKT and its downstream effectors in HER2+ breast cancer model

Evading apoptosis is considered to be a hallmark of cancers including breast cancer, since mutations in apoptotic regulators invariably accompany tumorigenesis. Chemotherapeutic agents induce apoptosis, and hence disruption of apoptosis during tumor progression may promote drug resistance. AKT is an apoptotic regulator that is activated in HER2+ breast tumor cells and promotes anti-HER2 therapy resistance in vitro . Nevertheless, how mTORC1/C2-AKT signaling disables apoptosis and its contribution to clinical drug resistance are not clear yet. Using HER2 amplified breast cancer cells [BT474 ( HER2+/ Trastuzumab-sensitive), BT474HerR ( HER2+/ Trastuzumab-resistant), HCC1954 and MDA-MB453 (both are HER2 +/ PIK3CA kinase domain mutated)], we show that mTORC1/C2 inhibitor; MLN0128 abrogates AKT (Ser473), Survivin and controls its downstream effectors of apoptotic signaling molecules (e.g. cleaved Caspase 3/9, cleaved PARP, MCL and BIM). MLN0128 also induces annexinV positive cells and regulates cellular proliferation (ON-TOP 3D colony formation and real-time proliferation assay). Additionally, increased cleaved Caspase 3 and decreased MCL1 expression were also observed following MLN0128 treatment in HER2+ xenograft model along with tumor growth inhibition. Our studies provide strong experimental evidence that high apoptotic signaling –specifically reduced MCL1 and increased cleaved-CASPASE3 expression expedite the response of targeted therapy that directly inhibits mTORC1/C2-AKT signaling. Citation Format: De PK, Carlson JH, Sun Y, Lin X, Williams C, Dey N, Leyland-Jones BR. MLN0128 regulates survival signaling by AKT and its downstream effectors in HER2+ breast cancer model. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-14.

Abstract P3-14-08: Preclinical efficacy of targeting c-MET by ARQ197 in combination with PARP inhibitor plus standard cytotoxic agent in triple-negative breast cancer cell lines

Background: Triple-negative breast cancer (TNBC), accounts for 15% of all invasive breast cancers (BCs) and has the poorest survival outcome of all BC subtypes. Due to its heterogeneity, TNBC lacks validated therapeutic targets compared with other BC subtypes (Sohn et al., 2014; Foulkes et al., 2010). Therefore, improved approaches to treatment of these cancers are unmet needed. Several molecular targets including: epidermal growth factor receptor (EGFR), poly ADP ribose polymerase (PARP), and hepatocyte growth factor receptor (c-MET) are under clinical investigation for the treatment of this disease (De et al., 2014; Cleator et al., 2007). The MET oncogene encodes a membrane-bound tyrosine kinase implicated in the formation and/or progression of several cancer types including TNBC, and several studies have shown c-Met overexpression to be an independent predictor of poor outcome in BC (Ho-Yen et al., 2014), c-MET may play a critical role in the development of the most aggressive BCs and may be a rational therapeutic target (Graveel et al., 2009). Currently inhibitors targeting c-MET (including ARQ197) are undergoing clinical trials in a variety of cancers including TNBC (Gaule et al., 2014; ClinicalTrials.gov). Recently, PARP inhibitors in combination with chemotherapy, has shown promising results in TNBC in clinical and preclinical studies (Tutt et al., 2010; De et al., 2014). We argue that, blocking the PARP-mediated nuclear machinery for repairing DNA-damage in presence of cytotoxic DNA damaging agents in conjunction with co-targeting c-Met pathway dependent downstream effectors may have a robust anti-tumor activity in TNBC cell lines. Methodology: BT-20 (PIK3CA mutated, H1047R), HCC70 (PTEN null), HCC1937 (PTEN null), MDA-MB-231 (KRAS/BRAF mutated), MDA-MB-468 (PTEN null) and SUM149PT (BRCA1 mutated) cells were used for this study. Growth inhibition, survival/proliferation, colony formation and apoptosis were examined using MTT assay, 2D proliferative /growth assay, 3D-ON-TOP assays, and annexinV staining respectively. Results: 1) For all TNBC cell lines, the IC50 of single agent ARQ197 was from 0.5 µM to 1.5 µM (following 96 hours treatment) 2) ARQ197 as a single agent or in combination with ABT888 or in triple combination dose dependently decreased cell growth/proliferation 3) annexin V positive cells were increased following treatment with single agent ARQ197 or in combination with ABT888 or in triple combination 4) 70-99% anti-proliferative activities were observed on 3D-ON TOP colony formation assay with ARQ197 alone or in combination in all tested cell lines. Conclusion: Our preclinical in vitro drug sensitivity data suggest that administration of c-MET inhibitor may enhance the antitumor activity of PARP inhibitor plus standard cytotoxic agent in TNBC models. Mechanism studies are ongoing, the results of which will be presented in the meeting. Citation Format: Sun Y, Carlson JH, Lin X, De PK, Williams C, Hausman S, Dey N, Leyland-Jones BR. Preclinical efficacy of targeting c-MET by ARQ197 in combination with PARP inhibitor plus standard cytotoxic agent in triple-negative breast cancer cell lines. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-08.

Abstract P4-08-04: Navigating genomic landscape to find a PI3K-signaling algorithm for a rational combinatin in precision medicine

Background: Treatment of BC is conventionally based on the presence/absence of ER/PR or HER2 status of the primary tumor. We have enriched this approach by including major genetic and proteomic changes in tumors of individual patients in order to develop a better treatment-rationale based on an alteration driven signaling algorithm. Methods: Genomic and proteomic data from 75 BC patients seen in our center were retrospectively analyzed. Patients were re-biopsied after consultation and samples were characterized (IHC for ER, PR, and HER2; FFPE samples for genomic [Foundation Medicine] and proteomic analyses [Theranostics]). In vivo studies were conducted using xenograft models. Results: Although alterations of PIK3CA, PIK3R1, AKT, PTEN, MDM2, MDM4, TSC1, mTOR and RICTOR are most frequently observed in our patients, there is a distinct pattern of alteration(s) of the PI3K pathway genes in different subtypes of BC. A total of 76 genes were altered in 48 ER+BC patients. In 79% of ER+BC patients the above mentioned PI3K pathway genes were altered. Analyzing the set of alterations of genes in individual patients, we observed that within these 48 patients 25% exhibited alterations in more than one node of the pathway; the most common combination (alterations) being the amplification/mutation of PIK3CA with the amplification of MDM2/4 genes. The percentage of patients belonging to HER2+ & TNBC exhibiting similar alterations in the PI3K pathway genes were significantly lower (∼40%). Our previous in vivo studies demonstrated that GDC-0980 and BEZ235 enhanced the antitumor activity of ABT888 plus carboplatin in TNBC or trastuzumab in HER2+ BC respectively and blocked the growth of established xenograft tumors by 80% to 90% with a concomitant decrease in tumor Ki67, pS6RP and CD31. Mechanistically the action of the PI3K-mTOR pathway targeted drug(s) was tested using cell line based models of BC subtypes pertaining to their respective genomic alterations. A combination of a pan-PI3K pathway inhibitor, GDC-0941 or isoform-specific inhibitors along with AI, trastuzumab, or HRD inhibitors (PARP) blocked proliferative signals and enhanced apoptosis (cleaved caspase3) in ER+/PIK3CA mutated, HER2+/PIK3CA mutated or PTEN-null TNBC cells respectively as demonstrated by WB, flow cytometry, cell proliferation, viability and cytotoxicity assays. A recent study demonstrated that exposure to chemotherapy induced a phenotypic shift or cell state transition towards a transient CD44Hi/CD24Hi chemotherapy-tolerant state, leading to the activation of downstream non-receptor tyrosine kinase signaling towards an emerging adaptive resistance (Goldman et al., Nature Comm. 2015). Hence drug combination(s) are being tested for their effect on CD44/CD24 expression levels, results of which will be presented in the meeting. Conclusion: Plotting the genetic alterations from the patient on the signaling landscape will be useful in cracking the code leading to improved treatment options. Patient specific in-depth plotting of genetic alterations of the PI3K-mTOR pathway and the relevance of these alterations in the context of (1) mechanisms of PI3K-mTOR pathway targeted drugs and (2) cell signaling are critical in determining choice of drugs in BC subtypes. Citation Format: Carlson JH, Krie A, Williams C, Sun Y, Lin X, Williams K, Klein J, Friedman L, De P, Dey N, Leyland-Jones B. Navigating genomic landscape to find a PI3K-signaling algorithm for a rational combinatin in precision medicine. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-08-04.

Abstract 5126: Does genetic background in glioma influence the sensitivity of tumor cells to a PARP inhibitor in the presence of different DNA damaging agents

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Upregulation of cellular signaling pathways for proliferation is one of the characteristic features of high-grade glioblastomas (GBM), the most common and lethal type of adult brain tumors. Common oncogenic alterations in GBM include a characteristic gain-of-function of growth-promoting EGFR gene, a functionally relevant activation of p21-RAS pathway or a loss-of-expression of growth-attenuating tumor suppressor PTEN gene. Here we tested the role of genetic backgrounds (RAS pathway upregulation and PTEN nullness) on the sensitivity of ABT888 in the presence of either temozolomide (TMZ) or carboplatin (C). Methods: We examined the anti-tumor effects of ABT888 using PTEN null U87MG cell line and a transgenic mouse model (12V-Ha-Ras transgenic mice) of human glioma expressing V12 RAS (Can. Res. 2001). LacZ, GFAP and PCNA-positive primary astrocytes (derivative astrocytes, passage 100) were used for the study. The effect of the drugs were tested by (1) soft agar colony growth (2) 3D- On-Top assay (3) cell cycle analyses (4) vascular mimicry (real time) and (5) Western blots in a dose and time dependent manner. Results: Expression of the transgene in 12V-Ha-Ras mice (tail biopsy of mice from which derivative glioma cells cultures were established) and in the derivative glioma cells (established from the transgene-expressing mice) at different passages were confirmed by genotyping. Testing for astrocytic marker GFAP by ICC (>95% GFAP positive at passage 10 and onwards). Both U87MG glioma cells and astrocytoma cells from 12V-Ha-Ras mice were positive for GFAP and PCNA. Our results show that (1) the combination of ABT888 plus TMZ is additive in reducing soft agar colony growth in both U87MG and V12RAS astrocytes (2) ABT888 sensitized both U87MG and V12RAS astrocytes to TMZ in 3D On-Top assay, (3) in both U87MG and V12RAS astrocytes the largest amount of G0 was observed following the combination of ABT888 plus C at 72 hours (4) ABT888 plus C as well as ABT888 plus TMZ blocked vascular mimicry in both U87MG and V12RAS astrocytes and (5) at 72 hours PTEN null U87MG cells exhibited an increase in pro-apoptotic cl-PARP and a decrease in anti-apoptotic MCL-1 following both combination treatments, an effect not observed in V12RAS astrocytes. At 48 hours the TMZ induced increase in PAR is blocked by ABT888, an effect more pronounced in U87MG cells. Conclusion: The data demonstrate that ABT888 sensitizes the effect of TMZ in PTEN null glioma cells. The functional link between tumor suppressor, PTEN and the sensitization of PARP inhibitor is being currently worked out utilizing glioma cells expressing PTEN to specifically address the role of PTEN nullness in the context of ABT888 in combination with TMZ, the results of which will be presented at the meeting. Citation Format: Jennifer H. Carlson, Brian Leyland-Jones, David Martin, Pradip De, Nandini Dey. Does genetic background in glioma influence the sensitivity of tumor cells to a PARP inhibitor in the presence of different DNA damaging agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5126. doi:10.1158/1538-7445.AM2014-5126

Abstract 4759: Integral role of the PI3K-AKT-mTOR pathway in DDR-mediated antitumor actions of PARP inhibitor in triple-negative breast carcinogenesis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: The PI3K pathway, in addition to its pro-proliferative and anti-apoptotic effects on tumor cells, is known to contribute to DNA-damage repair (DDR). We hypothesized that GDC-0980, a dual PI3K-mTOR inhibitor, would potentiate an anti-tumor effect in BRCA-competent TNBC cells when combined with the PARP inhibitor, ABT888 and carboplatin. Methods: Athymic mice bearing BRCA-competent TNBC xenograft tumors were treated with GDC-0980 alone or in combination with ABT888 and carboplatin. Mechanism-based in vitro studies (MTT, CelltiterGLO, flow cytometry-based live/dead cell, cell cycle analyses, anchorage independent soft-agar, anchorage dependent 3D ON-TOP, apoptosis, cell signaling markers assays) using a panel of 5-7 BRCA-wt /mutant TNBC cell lines were performed. Results: GDC-0980 treatment led to DNA damage (increased pgH2AX; WB, IF), gain in PAR and a subsequent sensitization of BRCA-competent TNBC cells to ABT888 plus carboplatin in a time-dependent manner. The treatment (1) decreased proliferation signals (pAKT, pP70S6K, p4EBP1, pS6RP), PAR/PARP, PAR/pgH2AX, live/dead cell ratios, cell cycle progression and clonogenic 3D growth, and (2) increased apoptosis markers (cl-caspase3, 9, BIM, cl-PARP, and annexinV positivity). These effects were more pronounced in PTEN-null MDA-MB468 than in RAS/RAF mutated MDA-MB231 cells. Three-dimensional projection movie showed that (1) GDC-0980 alone attenuated nuclear pγH2AXS139 foci in MDA-MB468 cells at 24 hours and (2) cytoplasmic cl-caspase3 increased in GDC-0980+ABT888+carboplatin treated MDA-MB468 cells at 72 hours. Finally, GDC-0980 in combination with ABT888 plus carboplatin blocked the growth of established xenograft tumors by 80-90% with a concomitant decrease in tumor Ki67, cl-caspse3, pVEGFR, CD31, p4EBP1, and pS6RP IHC-levels. Conclusion: This is the first mechanism-based study to demonstrate the inhibition of DDR as another mode of action of GDC-0980. Our data revealed that in a BRCA-competent model, GDC-0980 enhanced the antitumor efficacy of ABT888 in the presence of carboplatin by inhibiting the DDR system in conjunction with inhibition of the PI3K-mTOR pathway mediated pro-proliferative and anti-apoptotic signals. Citation Format: Brian Leyland-Jones, Jennifer Carlson, Yuliang Sun, Lori Friedman, Pradip De, Nandini Dey. Integral role of the PI3K-AKT-mTOR pathway in DDR-mediated antitumor actions of PARP inhibitor in triple-negative breast carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4759. doi:10.1158/1538-7445.AM2014-4759

Abstract 3443: Wnt-beta-catenin-Rac1 signaling axis regulates metastasis-associated phenotypes in TNBC

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: RAC1-GTPase controls many cellular phenotypes including integrin-directed migration via its control over the actin-cytoskeleton. We and others have established the upregulation of Wnt-beta-catenin pathway (WP) in triple negative breast cancer (TNBC), the most aggressive subtype of breast cancer (Dey et al., 2013, BMC Cancer; Dey et al., 2013, PLOS ONE). Here we report for the first time that metastasis-associated phenotypes in TNBC are controlled by Wnt-beta-catenin-RAC1signaling axis. Methodology: The involvement of WP was tested using (1) WP modulators (CHIR99021, Wnt-C59, XAV939), (2) sulindac sulfide and (3) beta-catenin SiRNA. We studied metastasis-associated phenotypes including, (1) proliferation (CelltiterGLO) (2) clonogenic growth (3D On-TOP assay) (3) fibronectin-directed migration (4) matrigel invasion (5) vascular mimicry (6) actin dynamics (confocal-microscopy and podia-parameters) (7) protein expression (WB) (8) real-time imaging for migration and vascular mimicry and (9) adhesion in the context of RAC1 activation in a panel of 3-4 TNBC cell lines. Results: Sulindac sulfide as well as transient transfection of beta-catenin SiRNA (1) decreased cellular levels of beta-catenin, inhibited proliferation, attenuated metastasis-associated different phenotypes and blocked fibronectin-mediated RAC1 as well as Cdc42 activities and (2) altered actin dynamics and inhibited podia-parameters (lamellopodia and fillopodia). Both Wnt-antagonists and RAC1 inhibitor (NSC23766) blocked fibronectin-induced RAC1 activation and inhibited the metastasis-associated phenotypes, while Wnt-activator enhanced phenotypes like vascular mimicry. Conclusion: Results of our experiments show that upregulation of WP regulates metastasis-associated phenotypes in TNBC via activation of RAC1 GTP-ase. The functional link between WP, metastasis and the activation of RAC1 is being currently worked out using RAC1SiRNA and pharmacological inhibitor of RAC1 in brain metastasizing MDA-MB231BR cells to specifically address the role of Wnt-beta-catenin-RAC1 signaling axis in the context of distant metastasis, the results of which will be presented at the meeting. Citation Format: Nandini Dey, Jennifer Carlson, Pradip De, Brian Leyland-Jones. Wnt-beta-catenin-Rac1 signaling axis regulates metastasis-associated phenotypes in TNBC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3443. doi:10.1158/1538-7445.AM2014-3443

Abstract 4223: Combined PI3K-AKT and MEK-ERK pathway inhibition provides broad antitumor efficacy in HER2+ breast tumor models

Background: The PI3K-AKT-mTOR and the RAS-RAF-MEK-ERK pathways are thought to be the central transducers of oncogenic signals in cancer. Combined targeting of these two pathways may be necessary for optimal therapeutic activity in solid tumors. This study evaluated the PI3K inhibitor GDC-0941, the MEK1/2 inhibitor GDC-0973 and the combination of both in different HER2+ breast cancer cell lines. Methodology: Growth inhibition/survival, apoptosis, signal transduction and antitumor efficacy were examined using MTT assays, 3D-ON-TOP assays, annexinV staining and mouse xenografts respectively, in BT474 (trastuzumab sensitive), BT474HR (trastuzumab-resistant) and HCC1954 (PIK3CA mutated) breast cancer cell lines. Results: (1) GDC-0941 and GDC-0973 exhibited in vitro cell killing activity in MTT assays with IC509s ranging from 0.35 µM to 1 µm and from 8 µM to 20 µM respectively, (2) the initiation of apoptotic activity (annexin V) in GDC-0941 plus GDC-0973 was significantly superior to that of either drug alone, (3) inhibition of phosphorylation of AKT(S473, T308), P70S6K, S6RP, and 4EBP1(T37/46) was observed following GDC-0941 treatment, and the combination of GDC-0941 plus GDC-0973 more effectively blocked p-S6RP and p-4EBP1, especially in BT474HR and HCC1954 cells, (4) GDC-0973 abrogated activation of ERK, and this ERK inactivation was sustained when combined with GDC-0941, (5) a combination of GDC-0941 and GDC-0973 more effectively blocked 3D-ON-TOP clonogenic growth of HER2+ cells than either agent alone, and (6) xenograft data show that the combination of GDC-0941 and trastuzumab has an enhanced anti-tumor effect in both sensitive (almost 100%) and resistant models (85%), and tumor regression was observed in both sensitive and resistant models when mice were treated with GDC-0941 plus GDC-0973 plus trastuzumab. Conclusions: Therapy with dual PI3K and MEK combination is more efficient than with either drug alone in all three cell lines. Our data provide stronger evidence that the addition of MEK inhibitor along with pan-PI3K inhibitor to HER2-directed treatment could augment the clinical benefit in breast cancer patients. Citation Format: Pradip De, Yuliang Sun, Jennifer Carlson, Lori Friedman, Nandini Dey, Brian Leyland-Jones. Combined PI3K-AKT and MEK-ERK pathway inhibition provides broad antitumor efficacy in HER2+ breast tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4223. doi:10.1158/1538-7445.AM2014-4223

Abstract 4231: Enhanced antitumor efficacy of T-DM1 in combination with pertuzumab in HER2-positive breast cancer models

Background: The HER2 oncogene is amplified in approximately 20-25% of human breast cancers and is a well established therapeutic target. HER2-targeted therapy by trastuzumab (T) has become increasingly important for treating HER2+ breast cancers, and following the EMILIA trial, T-DM1 is expected to serve as a suitable alternative to trastuzumab. Pertuzumab (P), a HER2 targeted monoclonal antibody that prevents HER2 dimerization, showed progression-free survival when used with trastuzumab and docetaxel on HER2+ metastatic breast cancer (CLEOPATRA trial). In this preclinical study, we investigated the effect of combining T-DM1 and P on xenografted HER2+ breast tumors. Methodology: Growth inhibition, apoptosis, and antitumor efficacy were examined using the 3D-ON-TOP assay, annexinV staining and mouse xenografts respectively, in BT474 (T- sensitive), BT474HR (T-resistant) and HCC1954 (PIK3CA mutated) breast cancer cell lines. Results: Treatment of HER2-overexpressing tumor cells (T-sensitive, T-resistant and PIK3CA mutant) in vitro with T-DM1 plus pertuzumab resulted in synergistic inhibition of cell proliferation (3D-ON-TOP and 2D clonogenic assays) and induction of apoptotic cell death (annexinV positive cells). The combination (T-DM1+P) also inhibited p-AKT (Ser473 and Thr308) in HER2-overexpressing cells. T-sensitive and T-resistant xenografts regressed completely after 6 weeks of therapy with T-DM1 plus P whereas either single agent inhibited tumor growth partially in both groups. Mice with HCC1954 (HER2+/PIK3CA mutated) xenograft treated with the same combination exhibited significant tumor growth inhibition when compared with vehicle treated mice after 6 weeks of treatment. Conclusions: Our in vitro and in vivo data clearly demonstrated that dual-targeting HER2 with a combination of T-DM1 plus P was highly efficacious both in HER2+/T-resistant and HER2+/PIK3CA mutated xenograft models. Citation Format: Yuliang Sun, Nandini Dey, Jennifer Carlson, Melissa Brammer, Pradip De, Brian Leyland-Jones. Enhanced antitumor efficacy of T-DM1 in combination with pertuzumab in HER2-positive breast cancer models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4231. doi:10.1158/1538-7445.AM2014-4231