Yuliang Sun

Abstract 4496: Preclinical efficacy of the novel dual PI3K/mTOR inhibitor, BEZ235 compared with RAD001 in HER2+ breast tumors

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: HER2 overexpression occurs in 20-25% of patients with breast cancer and is associated with aggressive disease and decreased survival. HER2 activates multiple cellular signaling pathways including the PI3K-AKT-mTOR pathway. The sustained activation of the PI3K-AKT-mTOR pathway or the activating mutation of PIK3CA (p110α) is one of the major causes of drug resistance including trastuzumab. Purpose: Inhibitors of mTOR1 have been shown to be active in patients with solid tumors. As the phosphatidylinositol 3-Kianse (PI3K) pathway activates numerous other kinases, transcription factors, and proteins associated with cell growth/survival and apoptosis besides mammalian target of rapamycin (mTOR), suppression of this pathway upstream of mTOR may be more effective than inhibition of mTOR1 in HER2+ breast cancer models. Experimental Design: To examine this possibility, the dual pan-PI3K/mTOR inhibitor, BEZ235 was compared with mTOR1 inhibitor, RAD001 in trastuzumab-sensitive HER2+ (BT474 and SKBR3), trastuzumab-resistant (BT474HR), and PIK3CA mutated/HER2+ breast cancer cells (HCC1954 and U893) Results: Treatment of HER2+ breast cancer cell lines with BEZ235 or RAD001 in vitro showed 1) RAD001 mediated AKT activation was blocked when cells were treated with BEZ235, 2) BEZ235 dose- and time-dependently blocked AKT phosphorylation (both at Ser473 and Thr308), 3) both drugs completely blocked the activation P70S6K, but BEZ235 more efficiently blocked 4EBP1 phosphorylation than RAD001, 4) ERK activation was significantly attenuated by BEZ235, while inhibition of mTOR1 by RAD001 lead to activated ERK, 5) BEZ235 was significantly more effective than RAD001 in reducing HIF1α expression following heregulin stimulation, 6) activation of caspase3 (from the expression level of cleaved caspase3) was also greater following treatment with BEZ235 than RAD001, and 7) integrin-dependent breast tumor cell migration was significantly abrogated with BEZ235 in association with inhibition of RAC1-GTP, but this was not seenwith RAD001. Conclusion: Our preclinical in vitro data demonstrate that concurrent inhibition of PI3K and mTOR is likely to be a more effective strategy in the treatment of both sensitive and resistant HER2+ breast cancers than the inhibition of mTOR1 alone. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4496. doi:10.1158/1538-7445.AM2011-4496

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

Abstract 545: In vitro potency of pan-inhibitor of class 1 PI3K, GDC0941 in ER+ and HER2 overexpressing breast cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Persistent activation of PI3K-AKT signaling and its regulated mTOR axis promote cell growth and proliferation in a variety of tumor types including breast tumors. A strong association has been demonstrated between mutational activation of PIK3CA or loss of function of PTEN, and resistance to therapies targeted against the ER or HER2 pathway. Purpose: Since PI3K-AKT-mTOR is one of the major signaling pathways responsible for the progression of breast cancer, and appears to be upregulated during development of the resistance to the endocrine and trastuzumab treatment, suppression of this pathway by administration of GDC0941 (PI3K inhibitor) may therefore be efficacious in ER+ and HER2+ breast cancer models. Experimental Design: GDC0941 was treated in ER+ (MCF7, HCC1500, BT483, T47D and MDA-MB-415), HER2+/ trastuzumab (T)-sensitive (BT474), T-resistant (BT474HR), and PIK3CA mutated/HER2+ breast cancer cells (HCC1954 and UACC893). We tested the effects of GDC0941 on the (a) cell survival/proliferation, (b) integrin-directed cell migration, (c) downstream signaling pathways for proliferation and apoptosis, and (d) hypoxia-induced HIF1α accumulation. Results: Treatment of GDC0941 in ER+ and HER2+ cell lines in vitro showed that 1) cell lines were sensitive to single agent GDC0941 with IC50 ranging from 0.3 μM to 2.5 μM in ER+ cell lines and 0.35 μM to1.0 μM in HER2+ cell lines, 2) GDC0941 dose- and time-dependently blocked downstream effectors of the PI3K-AKT pathway i.e. p-AKT (at Ser473, Thr 308), p-P70S6K, p-S6 ribosomal protein, and p-4EBP1(although in lesser extent) in all cell lines, 3) inhibition of AKT was more pronounced when T was combined with GDC0941 even at lower concentrations in both T-sensitive and T-resistant cells, 4) activation of ERK occurred in ER+/PIK3CA helical domain mutated, ER+PTEN-mutated cells, and HER2+/T-resistant cells, 5) in HER2+ cell lines, GDC0941treatment resulted in induction of apoptosis via cleavage of CASPASE3 in a dose-and time-dependent manner, 6) GDC0941 significantly abrogated hypoxia-induced HIF1α stabilization in ER+ and HER2+ cells. Interestingly, in GDC0941 treated cells, MG132 (proteosome inhibitor) restored the inhibitory effect of GDC0941 on HIF1α protein levels in hypoxic conditions, and 7) integrin-dependent cell migration was significantly abrogated with GDC0941 in ER+ and HER2+ (both in T-sensitive and T-resistant) cells. Conclusion: Our preclinical in vitro data demonstrate that GDC0941 inhibits cell proliferation and PI3K signaling in both ER+ and HER2+ breast cancer cells. A combination therapy with anti-estrogen+PI3K inhibitor and anti-HER2 agent+PI3K inhibitor significantly inhibits ER+ cell proliferation (including PIK3CA or PTEN mutated cells) and reverses HER2 resistance in our model systems. Citation Format: Pradip KR De, Yuliang Sun, Lori Friedman, Nandini Dey, Brian Leyland-Jones. In vitro potency of pan-inhibitor of class 1 PI3K, GDC0941 in ER+ and HER2 overexpressing breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 545. doi:10.1158/1538-7445.AM2013-545

Abstract 2240: Pre-clinical potency of INK128, a highly potent TORC1/2 kinase inhibitor in the HER2 amplified breast cancer model

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: The PI3K-AKT-mTOR pathway is commonly activated in breast cancers due to the amplification of receptor tyrosine kinase (HER2), the mutation of PIK3CA or the loss of expression/function of PTEN. About 20% of HER2+ breast cancer patients have PIK3CA mutation, which has resulted in a trend toward shorter progression-free survival with trastuzumab-based therapy. Resistance (intrinsic or acquired) to trastuzumab is a significant clinical challenge, partly due to the overriding activation of PI3K-AKT-mTOR pathway. The PI3K pathway activation leads to stimulation of the key growth and proliferation regulatory kinase, mTOR which can be blocked by allosteric inhibitors (rapalogous) or by ATP-comptetive mTOR kinase inhibitor (TORC1/2). Purpose: We investigated the effects of INK128; a potent and selective TORC1/2 kinase inhibitor alone or in conjunction with trastuzumab in HER2 amplified breast tumor cell lines. We also sought to identify the molecular response determinants of mTOR kinase inhibitor predicting potent activity in HER2+/trastuzumab-resistant cell lines. Experimental Design: Here, we used the HER2 amplified trastuzumab-sensitive breast tumor cell line (BT474), the trastuzumab-resistant breast tumor cell line (BT474HR) and HER2 amplified/PIK3CA mutated breast tumor cell lines (HCC1954, UACC893). Results: We demonstrate that 1) HER2+ cells are sensitive to INK128 (IC50 ∼40nM), 2) INK128 is also effective in trastuzumab-resistant and PIK3CA mutaed/HER2+ breast tumor cells (IC50 ∼20 and 20-30nM respectively), 3) INK128 is mechanistically distinct from rapalogous by a more potent and comprehensive inhibition of PI3K-AKT-mTOR signaling network: a) avoids S6K inhibition-mediated feedback activation of PI3K-AKT and b) abrogates 4EBP1 phosphorylation; 4) ligand-induced HIF1α accumulation is inhibited by prior treatment of INK128, and 5) the combination of trastuzumab plus INK128 more effectively blocked PI3K-AKT-mTOR signaling and HIF1α accumulation both in parental and trastuzumab-resistant cells. Conclusion: Our results suggest that i) therapeutic targeting of PI3K-AKT-mTOR signaling should be effective in abrogating resistance to trastuzumab therapy in HER2+ breast cancer, ii) combination of trastuzumab and INK128 is a promising treatment approach for HER2 overexpressed as well as trastuzumab-resistant breast cancer patients and iii) dual TORC1/2 inhibitor also mitigates the feedback activation of AKT caused by selective inhibition of mTORC1, and because of this, INK128 may yield greater therapeutic benefit in breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2240. doi:1538-7445.AM2012-2240

Abstract 2227: P110 α-specific inhibitor is more suitable in PIK3CA mutated breast cancer model but ineffective in PTEN loss of function breast cancer model

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Members of the phosphatidylinositiol 3-kinase (PI3K) pathway are frequently upregulated/amplified in broad range of cancers including breast cancer. The PIK3CA mutation or the loss-of-function of PTEN, which are found in different subsets of breast cancer, activates the PI3K-AKT-mTOR pathway, resulting in breast cancer development, progression and lower clinical benefit rate. Purpose: We investigated the comparison of inhibitory potency of p110α-specific inhibitor (INK1402) versus PI3K/mTOR dual inhibitor (BEZ235) in PI3K activated (define as activating mutation of PIK3CA or by loss-of-function of PTEN) hormone receptor positive (HR+) and triple negative (TN) breast tumor cells (a proof-of-concept study). Experimental Design: We used HR+ (MCF7, T47D and MDA-MB415) and TN (MDA-MB231 and MDA-MB468) breast cancer cell lines. We have tested the effects of INK1402 and BEZ235 on the, (a) cell survival/proliferation, (b) downstream signaling pathways for proliferation and (c) 3D ON-TOP-colony formation. Results: Here, we demonstrate that 1) both PIK3CA mutated and PTEN null or mutated breast cancer cells (ER+ and TN) are sensitive to BEZ235 (∼IC50 7 nM-30 nM), 2) only PIK3CA mutated cells (not PTEN loss-of-function cell lines) are sensitive to INK1402 (∼IC50 2 µM-12 µM), 3) anti-clonogenic growth property (3D-ON TOP colony formation assay) of INK1402 is more pronounced (80%-100%) in PIK3CA mutated cells as compared to PTEN loss-of-function cells (∼50%-60%), 4) BEZ235 shows anti-clonogenic property (3D-ON TOP colony formation assay) in all cell lines (∼60%-80%), 5) INK1402 and BEZ235 differentially inhibit PI3K-AKT-mTOR pathway and RAS-MAPK pathway in PIK3CA mutated, PTEN mutated (ER+), and PTEN null (TN) breast cancer cell lines: a) INK1402 is significantly more effective in PIK3CA mutated cell lines as compared to PTEN null or mutated cell lines, b) BEZ235 blocks the activation of PI3K-mTOR pathway components (p-AKT, p-S6RP) in both PIK3CA mutated and PTEN null or mutated cell lines, and c) interestingly, INK1402 blocks ERK activation in PIK3CA mutated cells whereas BEZ235 upregulates ERK activation in both PI3K mutated and PTEN null or mutated cell lines. Conclusion: These findings confirm the mutually exclusive behavior of PIK3CA mutation and PTEN loss in breast tumor models (Ellis MJ et al Cancer Research 2009; Russillo M et al JCO 2011). Our initial finding may provide a rationale to guide selection of patients who may benefit from PI3K-isoform (p110α)-specific inhibitor or PI3K/mTOR dual inhibitor therapy, and highlights the importance of accurately accessing the expression/functional status of PIK3CA or PTEN status in breast tumor samples. Acknowledgement: We sincerely thank to Novartis and Intellikine Inc for proving us with BEZ235 and INK1402 respectively. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2227. doi:1538-7445.AM2012-2227

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.

Down’s Syndrome and Triple Negative Breast Cancer: A Rare Occurrence of Distinctive Clinical Relationship

Down’s syndrome (DS), the most common genetic cause of significant intellectual disability in children and adults is caused by the trisomy of either all or a part of human chromosome 21 (HSA21). Patients with DS mostly suffer from characteristic tumor types. Although individual patients of DS are at a higher risk for acute leukemia and testicular cancers, other types of solid tumors including breast cancers are mostly uncommon and have significantly lower-than-expected age-adjusted incidence rates. Except for an increased risk of retinoblastomas, and lymphomas, the risk of developing solid tumors has been found to be lower in both children and adults, and breast cancer was found to be almost absent (Hasle H., The Lancet Oncology, 2001). A study conducted in the United States found only one death when 11.65 were expected (Scholl T et al., Dev Med Child Neurol. 1982). A recent study examined mammogram reports of women with DS treated in the largest medical facility specifically serving adults with DS in the United States. It was found that only 0.7% women with DS had been diagnosed with breast cancers (Chicoine B et al., Intellect Dev Disabil. 2015). Here we describe a case of breast cancer in a 25-year-old patient with DS. The disease was presented as lymph node positive carcinoma with alterations of tumor suppressor genes characteristic to the triple negative breast cancer subtype. Comprehensive Genomic Profiling (CGP) revealed a wild-type status for BRCA1. The CGP report showed a frameshift mutation, A359fs*10 of the tumor suppressor gene INPP4B and another frameshift mutation, R282fs*63 of tumor suppressor gene TP53 in the tumor biopsy as characteristically found in triple-negative breast cancers. The VUS (Variance of Unknown Significance) alteration(s) were identified in ASXL1 (L1395V), NTRK1 (G18E), DDR2 (I159T), RUNX1 (amplification), ERG (amplification), SOX2 (T26A), FAM123B (G1031D), and HNF1A (A301T). Bonafide cancer-related genes of chromosome 21 amplified in the patient’s tumor are RUNX1 and ERG genes. After the completion of the radiation, the patient was placed on everolimus which was based on the result of her CGP report. Thus, post-mastectomy radiation therapy was completed with a recommendation for everolimus for one year. During the time of writing of this report, no metastatic lesions were identified. The patient currently has no evidence of disease.

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 1348: GDC-0032, a p110 beta sparing PI3K inhibitor is highly efficient on PIK3CA-mutated and HER2-amplified breast cancer model

Background: HER2 amplification/overexpression frequently co-occurs with PI3K pathway activation in breast tumors. Mutations in PIK3CA are known to be involved in a wide range of human cancers including HER2+ breast cancer, and mutant PIK3CA is thought to act as an oncogene. Here, we evaluate the efficacy of GDC0032, a p110 beta-sparing PI3K inhibitor against HER2+ or HER2+/PIK3CA mutated breast cancer cells. MATERIALS AND METHODS: Four HER2+ breast cancer (BC) cell lines consisting of three activating (either helical or kinase domain) PIK3CA mutation (HER2+/ER+ and HER2+/ER-) were analyzed for proliferation, apoptosis, cell cycle and signaling pathway activation assays. Preclinical efficacy of GDC-0032 was also evaluated in vivo in a mouse model. RESULTS: 1) All HER2+ and HER2+/PIK3CA mutant cell lines exhibited low IC50 values (ranging from 0.1 µM-1.5 µM) irrespective of ER-positive or not. 2) GDC-0032 caused a strong differential growth inhibition in both HER2+ and HER2+/ /PIK3CA mutated breast cancer cell lines when compared to lines that were HER2- and PIK3CA wild type by 3D-ON-TOP clonogenic assay. 3) Administration of GDC-0032 induced cell cycle G0/G1 arrest and resulted in increased apoptosis in a dose-dependent manner. Furthermore, induction of apoptosis was more with GDC-0032 when combined with RAD001. 4) GDC0032 also blocked expression of CYCLIN D1. 5) Investigation of the signal transduction revealed that the treatment of GDC-0032 reduced the level of p-AKT (Ser473 and Thr308), and p-S6 expression is indicating the down-regulation of downstream signaling of PI3K and mTOR pathway. 6) GDC-0032 was highly active at reducing established tumor growth in vivo in BC mouse xenografts harboring PIK3CA mutation and HER2 amplification. CONCLUSION: Our in vitro or in vivo data showed that GDC-0032 was highly efficient either as a single agent as well as with RAD001 in HER2+/PIK3CA mutated breast cancer cell lines. Our data suggest that GDC-0032 represents a novel therapeutic option in patients harboring PIK3CA mutations and/or HER2/neu gene amplification in breast cancer. Citation Format: Pradip De, Yuliang Sun, Jennifer Carlson, Lori Friedman, Casey Williams, Nandini Dey, Brian Leyland-Jones. GDC-0032, a p110 beta sparing PI3K inhibitor is highly efficient on PIK3CA-mutated and HER2-amplified breast cancer model [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 1348. doi:10.1158/1538-7445.AM2017-1348

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.