Xiaoqian Lin

Abstract 1940: Dual inhibition of mTORC1/C2 and HER2 results in maximal antitumor efficacy in preclinical model of HER2+ breast cancer resistant to trastuzumab therapy

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Approximately 30% of HER2+ breast cancers (BC) harbor activating mutations (helical or kinase domain) in PIK3CA (Ellis and Perou Cancer Discovery 2013 3:27) and PIK3CA mutation is associated with lower rates of pathologic complete response to anti-HER2 therapies in primary HER2+ BC (Loibl S et al JCO 2014 55:7876). The hypothesis is that the suppression of one of the nodal points of this pathway results in sensitization to anti-HER2 agents. However, the combination of trastuzumab (T) and mTORC1/C2 inhibitor has not been comprehensively tested in T-sensitive and HER2+/PIK3CA mutated models. Methodology: Here we investigate the preclinical efficacy of a dual mTORC1/C2 inhibitor (MLN-0128, formally called INK128) alone or in combination with T in HER2+/T-sensitive/PIK3CA wild type (neither helical nor kinase domain mutation) (BT474), and HER2+/PIK3CA mutated (HCC1954, UACC893, MDA-MB453) cell lines. We analyzed in vitro cell viability and induction of apoptosis in all HER2+ cell lines. PI3K signaling was examined by immunoblot for phosphoproteins in HER2-PI3K-AKT-mTOR signaling cascade. Tumor growth inhibition was evaluated after the treatment with T, MLN-0128 or the combination in HER2 amplified models with wild type or mutant PIK3CA. Results: Our data showed that 1) PIK3CA mutation in HER2+ cells were responsible for resistance to the HER2-specific antibody T, 2) T-resistance conferred by this activating PIK3CA mutation was overcome by blockade of mTORC1/C2 with MLN-0128, 3) inhibition of phosphorylation of AKT(S473), P70S6K, S6RP, and 4EBP1(T37/46, T70) was observed following MLN-0128 treatment and the combination of MLN-0128 and T more effectively blocked the mTORC1/C2 signaling pathway, 4) MLN-0128 induced apoptosis of both BT474 and HCC1954 cells as indicated by Annexin V staining, 5) in vivo, both cell line-based xenografts showed exquisite sensitivity to the antitumor activity of the combination of T and MLN-0128, which resulted in durable tumor shrinkage and exhibited no signs of toxicity in these models and 6) immununo-staining of tumor tissues from MLN-0128 treated mice showed growth inhibition (Ki67), tumor-induced angiogenesis (CD31) and a decreased in associated phospho-proteins (p-S6RP, p-4EBP1). Conclusion: These results suggest that the combination of MLN-0128 plus T may be a potential strategy for the treatment of T-resistant breast cancer mediated by activating mutation of PIK3CA and this combination also provides benefit to HER2+/PIK3CA wild type tumors. Citation Format: Pradip Kr De, Yuliang Sun, Jennifer H. Carlson, Xiaoqian Lin, Nandini Dey, Brian Leyland-Jones. Dual inhibition of mTORC1/C2 and HER2 results in maximal antitumor efficacy in preclinical model of HER2+ breast cancer resistant to trastuzumab therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1940. doi:10.1158/1538-7445.AM2015-1940

High Expression of FGD3, a Putative Regulator of Cell Morphology and Motility, Is Prognostic of Favorable Outcome in Multiple Cancers

Purpose Identification of single-gene biomarkers that are prognostic of outcome can shed new insights on the molecular mechanisms that drive breast cancer and other cancers. Methods Exploratory analysis of 20,464 single-gene messenger RNAs (mRNAs) in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) discovery cohort indicates that low expression of FGD3 mRNA is prognostic for poor outcome. Prognostic significance of faciogenital dysplasia 3 (FGD3), SUSD3, and other single-gene proliferation markers was evaluated in breast cancer and The Cancer Genome Atlas (TCGA) cohorts. Results A meta-analysis of Cox regression of FGD3 mRNA as a continuous variable for overall survival of estrogen receptor (ER)–positive samples in METABRIC discovery, METABRIC validation, TCGA breast cancer, and Combination Chemotherapy in Treating Women With Breast Cancer (E2197) cohorts resulted in a combined hazard ratio (HR) of 0.69 (95% CI, 0.63 to 0.75), indicating better outcome with high expression. In the ER-negative samples, the combined meta-analysis HR was 0.72 (95% CI, 0.63 to 0.82), suggesting that FGD3 is prognostic regardless of ER status. The potential of FGD3 as a biomarker for freedom from recurrence was evaluated in the Breast International Group 1-98 (BIG 1-98; Letrozole or Tamoxifen in Treating Postmenopausal Women With Breast Cancer) study (HR, 0.85; 95% CI, 0.76 to 0.93) for breast cancer–free interval. In the Hungarian Academy of Science (HAS) breast cancer cohort, splitting on the median had an HR of 0.49 (95% CI, 0.42 to 0.58) for recurrence-free survival. A comparison of the Stouffer P value in five ER-positive cohorts showed that FGD3 (P = 3.8E-14) outperformed MKI67 (P = 1.06E-8) and AURKA (P = 2.61E-5). A comparison of the Stouffer P value in four ER-negative cohorts showed that FGD3 (P = 3.88E-5) outperformed MKI67 (P = .477) and AURKA (P = .820). Conclusion FGD3 was previously shown to inhibit cell migration. FGD3 mRNA is regulated by ESR1 and is associated with favorable outcome in six distinct breast cancer cohorts and four TCGA cancer cohorts. This suggests that FGD3 is an important clinical biomarker.

Abstract 3926: Is FGD3 a potentially prognostic marker for breast cancer

Background: Prognostic factors are capable of providing information on clinical outcomes at the time of diagnosis; they are usually indicators of growth, invasion, and metastatic potential (Gasparini G. et al. 1993; Hayes DF. et al. 1998). Tissue marker is one of the prognostic factors; to date, only a small proportion of markers are ultimately clinically useful, including Ki-67 and HER2. Faciogenital dysplasia 3 protein (FGD3), a putative regulator of cell morphology and motility, has been shown to regulate cell migration. In a study of 3,256 tumors, low expression of FGD3 mRNA indicates poor outcome (Hayakawa M. et al. 2008; Scooter W. et al. 2014). However, an immunohistochemistry (IHC) study to evaluate FGD3 protein expression has not been done. We hypothesize that the expression levels of FGD3 protein by IHC might improve the prediction of patient outcomes, and FGD3 might be a potentially prognostic marker of breast cancer. Materials and Methods: 322 cases of breast cancer Tissue Microarrays (TMA) (BR1504a, BR1505b, HBre-Duc068Bch-01, and BR20837) were purchased from US Biomax, Inc (Rockville, MD). Rabbit polyclonal antibody against FGD3 was purchased from Novus Biologicals, LLC (Littleton, CO). Tissue cores were stained for FGD3 with Dako’s EnVision + Dual Link System. Image acquisition was performed using an Olympus camera and software. FGD3 protein expression by IHC was quantitatively determined in the range of 0-4. Unpaired t-test was used for data analyses. Results: 1) Benign tumor and breast adenocarcinomas in lower stages showed strong expression of FGD3, whereas the breast adenocarcinomas in higher stages showed mild ~ weak expression. 2) Invasive breast cancer in stage IIA showed strong FGD3 expression compared to matched metastatic carcinoma which showed mild expression of FGD3. 3) FGD3 protein levels for tumors (n=135) with N0 indicating no regional lymph node metastasis were compared with tumors with lymph node metastasis (N1-3; n=98) and corresponding matched metastatic tissue (n=103). An unpaired t-test comparing N0 vs. N1-3 showed lymph node metastasis is associated with lower FGD3 protein levels (p Conclusion: FGD3 protein expression levels within breast tumors were different according to metastatic status. Our IHC results suggest the possibility of FGD3 to be a prognostic marker in patients with breast cancer. Citation Format: Yuliang Sun, Scooter Willis, Xiaoqian Lin, Justin Achua, Casey Williams, Brian Leyland-Jones. Is FGD3 a potentially prognostic marker for breast cancer [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 3926. doi:10.1158/1538-7445.AM2017-3926

Abstract B11: A dual PI3K/mTOR inhibitor, BEZ235 blocks tumor-induced angiogenesis: Evidence for an effect on the tumor and endothelial compartment

Abstracts: AACR Special Conference: Advances in Breast Cancer; October 17-20, 2015; Bellevue, WA Background: Tumor progression including HER2+ breast tumor, particularly in aggressive and malignant tumors, is associated with the angiogenesis. To investigate the effects of BEZ235 on angiogenesis, we evaluated its effects on the hypoxic stabilization or the PI3K-AKT-mTOR pathway-mediated synthesis of hypoxia-inducible factor -1α (HIF-1α) and tumor-induced angiogenesis. Methodology: Here we have studied the in vitro and in vivo effects of BEZ235 in HER2+/T-sensitive (BT474), HER2+/T-resistant (BT474HerR) and HER2+/PIK3CA (HCC1954 & UACC893) mutated models. We assessed in vitro activation status of the PI3K-AKT-mTOR signaling pathway following BEZ235 treatment in HER2+ BC cell lines. We next evaluated the impact of BEZ235 on hypoxia-induced or the PI3K-AKT-mTOR pathway ⊠mediated HIF1α stabilization/synthesis which is a master regulator of angiogenesis and also tumor-induced angiogenesis using xenograft models. Results: The results show that 1) BEZ235 inhibited downstream activation of the PI3K-AKT-mTOR signaling pathway effectors, p-AKT (Ser473, The308), p-P70S6K, p-S6RP and p-4EBP1, 2) unlike other pan-PI3Kinase inhibitor (e.g. LY294002, SF1126), BEZ235 has no effect on hypoxia-mediated stabilization of HIF1α however, heregulin-induced HIF1α synthesis which is an important angiogenic modulator in breast cancer cells, was significantly decreased following the treatment of BEZ235 in HER2+ and HER2+/PIK3CA mutated breast cancer cells, 3) interestingly, BEZ235 mediated abrogation of HIF1α synthesis is more pronounced than mTORC1 inhibition alone, 4) integrin-directed endothelial cell migration is one of the critical steps for tumor-induced angiogenesis. BEZ235 abrogates endothelial cells functions especially integrin-directed HUVEC cells migration on vitronectin (avb3/ avb5) and significantly abrogates endothelial cells tube formation on Matrigel and 5) furthermore, we evaluated the effects of BEZ235 on the capacity of HER2+ and HER2+/PIK3CA mutated breast tumor cells to recruit a blood supply in vivo during thrice weekly treatments with BEZ235. A microvessel density (MVD) in control versus BEZ235-treated tumors showed a significant decrease in MVD (CD31+) and also VEGFR immunostaining in BEZ235 treated tumors. Conclusion: Our data suggest that BEZ235 has potent antiangiogenic activity in three different xenograft models probably via mTOR-HIF1α-VEGF signaling axis. Citation Format: Pradip De, Yuliang Sun, Jennifer H. Carlson, Xiaoqian Lin, Nandini Dey, Brian Leyland-Jones. A dual PI3K/mTOR inhibitor, BEZ235 blocks tumor-induced angiogenesis: Evidence for an effect on the tumor and endothelial compartment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B11.

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 4417: In vitro efficacy of dasatinib alone or in combination with PARP inhibitor plus standard cytotoxic agent in triple-negative breast cancer cell lines

Background: Patients presenting with triple-negative breast cancer (TNBC) have the poorest prognosis of all breast cancer (BC) subtypes and there are limited treatment options, with no efficacious targeted therapies. Therefore, improved approaches to treatment of these cancers are an unmet need. Several molecular targets including: epidermal growth factor receptor (EGFR), poly ADP ribose polymerase (PARP), and Src family tyrosine kinases are under clinical investigation for the treatment of this disease (De et al., 2014; Kim et al., 2013). Src family tyrosine kinases play a critical role in signal transduction downstream of growth factor receptors and integrin family members. Preclinical studies have established a role for Src family kinases in proliferation, angiogenesis and invasion in prostate cancer model (Varcaris et al., 2014). Dasatinib is an rally-active ATP-competitive small molecule kinase inhibitor that potently inhibits Abl kinase, Src family kinases and other kinases (Lombardo et al., 2004), and has shown its anti-proliferative and anti-metastatic effectiveness against TNBC in both preclinical and clinical studies (Finn et al., 2011). Recently, PARP inhibitors in combination with chemotherapy have shown promising results in TNBC in clinical and preclinical studies (Tutt et al., 2010; De et al., 2014). We hypothesize that the inhibitor of Src family of kinases (dasatinib) in combination with PARP inhibitor (ABT888) plus standard cytotoxic agent (carboplatin) will attenuate the growth of 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, PTEN null) 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: Our data showed that 1) High anti-proliferative activities were observed following the treatment of dasatinib along with ABT888 plus carboplatin in both 2D proliferation assay and 3D-ON TOP colony formation assay. 2) In the same line, dasatinib in combination with ABT888 plus carboplatin inducing early stage apoptosis was seen by Annexin V staining in all tested cell lines. 3) Our MTT data showed that anti-proliferative activity of dasatinib as a single agent is highly variable in different genetic background of TNBC cell lines. Conclusion: Our data suggest that the combination of Src inhibitor may enhance the antitumor activity of PARP inhibitor plus standard cytotoxic agent in TNBC models. Mechanistic studies are ongoing, the results of which will be presented in the meeting. Citation Format: Yuliang Sun, Jennifer Carlson, Xiaoqian Lin, Pradip De, Casey Williams, Sally Hausman, Nandini Dey, Brian Leyland-Jones. In vitro efficacy of dasatinib alone or in combination with PARP inhibitor plus standard cytotoxic agent in triple-negative breast cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4417.

Abstract B107: Targeting PI3K and mTOR with GDC-0980, a dual inhibitor, is efficacious in trastuzumab refractory and HER2+/PIK3CA-mutated breast cancer models

Background: The PI3K-AKT-mTOR signaling plays an important role in breast cancer (BC). Its interaction with HER2 signaling becomes more complex and inter-dependent with acquired anti-HER2 therapy resistance. Targeting mTOR combined with endocrine therapy has shown clinical utility, however, a negative feedback-loop exists downstream of PI3K-AKT-mTOR. Furthermore, BOLERO 1 study clearly showed that PFS was not significantly different between groups (combination of everolimus with trastuzumab (T) plus paclitaxel versus placebo with T plus paclitaxel, 14.95 versus 14.49 months) in the full analysis population. Dual blockade of PI3K/mTOR is therefore an attractive drug for HER2+ BC, especially in T-resistant and PIK3CAmutated conditions. Methodology: Here we have studied the in vitro and in vivo effects of GDC-0980 in HER2+/T-sensitive (BT474), HER2+/T-resistant (BT474HerR), HER2+/PIK3CA (HCC1954, MDA-MB453) and HER2+/PTEN (HCC1569) mutated models. We assessed in vitro anti-proliferative, pro-apoptotic and activation status of the PI3K-AKT-mTOR signaling pathway following GDC-0980 treatment in HER2+ BC cell lines. We next evaluated the impact of GDC-0980 on tumor growth and angiogenesis using xenograft models. Results: 1) GDC-0980 inhibited downstream activation of the PI3K-mTOR signaling pathway effectors, p-AKT (Ser473, The308), p-P70S6K, p-S6RP and p-4EBP1, 2) the anti-proliferative activity of GDC-0980 was observed by 3D-ON-TOP clonogenic assay, 3) consistent with anti-proliferative effects of GDC-0980, the proportion of cells in the G1 phase of the cell cycle increased in all three cell lines with a concomitant decrease in the S phase of their treatment with GDC-0980, 4) the initiation of apoptotic activity (annexin V) of GDC-0980 was significantly superior to that of an allosteric inhibitor of mTOR, RAD001. GDC-0980 also induced apoptotic markers like cleaved CASPASE3, cleaved PARP1, BIM in BT474, BT474HerR & HCC1954 BC cells and 5) in the HER2+/T-sensitive, HER2+/T-resistant and HER2+/PIK3CA mutated BC xenograft models, GDC-0980 inhibited PI3K signaling and had potent anti-tumor activity. Along with its anti-tumor effect, GDC-0980 effectively decreased tumor angiogenesis (tumor micro-vessel density via CD31 staining). These inhibitions were more pronounced when GDC-0980 was combined with T. Conclusions: GDC-0980 inhibits the PI3K-AKT-mTORC1/C2 signaling pathway and results in anti-proliferative and anti-tumor activity in HER2+ breast cancer cells with both wild type and mutated PIK3CA. Citation Format: Pradip De, Yuliang Sun, Jennifer H. Carlson, Xiaoqian Lin, Friedman Lori, Nandini Dey, Brian Leyland-Jones. Targeting PI3K and mTOR with GDC-0980, a dual inhibitor, is efficacious in trastuzumab refractory and HER2+/PIK3CA-mutated breast cancer models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B107.