Y Sun

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 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.

Erratum to “Doubling Down on the PI3K-AKTmTOR Pathway Enhances the Antitumor Efficacy of PARP Inhibitor in Triple Negative Breast Cancer Model beyond BRCA-ness” [Neoplasia 16 (2014) 43–72]

[This corrects the article DOI: 10.1593/neo.131694.].

Abstract P3-04-02: Absence of PTEN facilitates the anti-tumor efficacy of GDC-0980 in combination with ABT888 plus carboplatin in BRCA1-competent triple negative breast cancer

Introduction: 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, will induce an efficient anti-tumor effect in BRCA-competent PTEN-null TNBC cells when combined with PARP inhibitor, ABT888 and carboplatin. We propose that in PTEN-null BRCA-competent TNBC model, the growth of TNBC tumor will be blocked due to the inhibition of (1) HR and NHEJ and (2) PI3K-mTOR pathway mediated survival signals following treatment with GDC-0980, when combined with PARP inhibitor (impaired DNA-SSB-repair) and carboplatin (increased DNA-DSB). Purpose: Here we tested the efficacy of a combination of GDC-0980 with ABT888 plus carboplatin in BRCA-competent PTEN-null model of TNBC. Methods: Athymic mice bearing PTEN-null TNBC xenograft tumors were treated with GDC-0980 alone or in combination with ABT888 and carboplatin. Results : Dual inhibition of PI3K and mTOR by GDC-0980 alone as well as in the presence of carboplatin plus ABT888 changed the state of the repair of DNA-damage in BRCA-competent PTEN null TNBC cells, which led to increased cellular apoptotic signals in addition to decreased survival/proliferative signals. GDC-0980 treatment led to DNA damage (increased pgH2AX), gain in PAR and a subsequent sensitization of BRCA-competent PTEN-null MDA-MB468 TNBC cells to ABT888 plus carboplatin with a time-dependent (1) decrease in proliferation signals (pAKT T308/S473, pP70S6K, pS6RP), PAR/PARP ratios, PAR/pgH2AX ratios, live/dead cell ratios, cell-cycle progression and clonogenic 3D growth and (2) increase in apoptosis markers (cleaved-caspase 3, 9, BIM, cleaved-PARP and annexinV positivity). These effects are more pronounced in MDA-MB468 than in RAS/RAF mutated MDA-MB231 cells. GDC-0980 alone and in combination with ABT888 plus carboplatin inhibited cell cycle progression, increased apoptosis, and decreased live/dead cell ratios in BRCA-competent PTEN null TNBC cells. GDC-0980 alone and in combination with ABT888 plus carboplatin attenuated anchorage -dependent and -independent clonogenic 3D growth comparatively more in BRCA-competent PTEN-null cells TNBC cells than MDA-MB231 cells. GDC-0980 in combination with ABT888 plus carboplatin blocked the growth of established PTEN-null TNBC tumors as compared to vehicle control(s) with a concomitant decrease in tumor Ki67 and CD31 IHC-stains. Conclusion : This is the first mechanism-based study to demonstrate that in BRCA-competent PTEN-null TNBC model, GDC-0980 enhanced antitumor activity of ABT888, in the presence of carboplatin by inhibiting DDR system in conjunction with the inhibition of PI3K-mTOR pathway-mediated proliferative, and anti-apoptotic signals. Considering (1) the importance of PARP as the target in TNBC, (2) the existence of a large percentage of BRCA-competent TN and/or basal type BC patients and (3) the high frequency of PTEN-null-ness in this subset of BC, this combination merits further investigation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-02.

Abstract P5-06-01: The PI3K inhibitor GDC-0941 combines with trastuzumab for superior anti-tumor efficacy in HER2+ breast cancer models

Background : PI3K-AKT-mTOR pathway signaling is important for the oncogenic function of HER2. Activating alterations of this pathway are frequently observed in HER2-enriched breast cancer and generally herald a poor response and resistance to trastuzumab (T). Purpose : Targeting the PI3K-AKT-mTOR pathway is an attractive strategy in HER2+ breast cancer that is refractory to trastuzumab. The hypothesis is that the suppression of this pathway by pan-PI3K inhibitor, GDC-0941 may lead to overcome trastuzumab-resistance. Experimental Design : The antiproliferative and HER2-mediated cellular signaling (pAKT, pP70S6K, pS6RP, p4EBP1 and p-ERK) effects of GDC-0941 alone and in combination with T were evaluated in HER2 amplified T-sensitive (BT474), T-resistant (BT474HR), and HER2 amplified/ PIK3CA mutated (HCC1954, UACC893) BT cell lines by MTT assay and Western blots. Clonogenic growth was tested by 3D ON-TOP assay and apoptosis markers were also tested. Athymic mice bearing BT474 and BT474HR xenograft tumors were treated with GDC-0941 and T (alone and in combination). Results : (1) GDC-0941 exhibited in vitro cell killing activity in MTT assay with IC509s ranging from 0.35 μM to 1 μm and potency was augmented by the addition of T, (2) inhibition of phosphorylation of AKT(S473, T308), P70S6K, S6RP, and 4EBP1(T37/46, T70) was observed following GDC-0941 treatment, and the combination of GDC-0941 and T more effectively blocked the PI3K-AKT-mTOR pathway, (3) GDC-0941 treatment increased apoptosis markers (CL-CASPASE3 and annexinV positivity), (4) GDC-0941 dose-dependently blocked 3D-ON-TOP clonogenic growth of HER2+ cells. This effect was potentiated in the presence of T and (5) in vivo, the combination of GDC-0941 and T significantly reduced established tumor growth in both sensitive (82%) and resistant (79%) models with concomitant decrease of different PD markers. Conclusions: Our data suggest that 1) therapeutic targeting of the PI3K-AKT-mTOR signaling should be effective in abrogating resistance to T therapy in HER2+ BT, and 2) targeting both the HER2 and the PI3K signaling pathways is an attractive strategy to enhance the clinical efficacy of T therapy, as well as to prevent or delay the development of resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-06-01.

Abstract P5-19-03: Olaparib plus carboplatin in combination with vandetanib inhibited the growth of BRCA-wt triple negative breast tumors in mice: Outside BRCA-box

Introduction: PARP inhibition has emerged as one of the most exciting and promising ‘targeted’ therapeutic strategies in the treatment of advanced triple negative breast cancer (TNBC) – the intended ‘target,’ being DNA repair ( Anders CK, 2010 ). Although olaparib is known to have antitumor activity in BRCA-related TNBC cells, a limited number of preclinical and clinical studies have shown antitumor efficacy of olaparib in non-BRCA-related BC ( Shimo T, 2012 ). Understanding the biology of TNBC cells has identified molecular targets including RTK(s), such as EGFR. Purpose: Here we tested the combination of a PARP inhibitor, olaparib (O) (AstraZeneca) plus carboplatin (C) with the EGFR/VEGFR inhibitor, vandetanib (V) (AstraZeneca) in a BRCA-wt TNBC model. Methods: Athymic mice bearing TNBC ( BRCA-wt VEGFR expressing MDA-MB231& MDA-MB468 [EGFR amplified/overexpressed]) xenograft tumors (200 mm 3 ) were treated with O plus C in combination with V (Arms: vehicle control 1, vehicle control 2, C+O, V, C+O+V). In vitro effects of V in combination with O plus C (or temozolomide) on clonogenic growth (3D on-TOP assay), proliferation (MTT and CelltiterGLO), apoptosis (Apoptosis Array), cell signaling marker(s) (Western Blot), and tumor cell phenotypes (fibronectin-directed migration, matrigel-invasion, and vascular mimicry) were investigated in a panel of five BRCA-wt and BRCA-mutated TNBC cell lines. The effects of V were tested on (a) cell signaling marker(s), (b) angiogenesis marker (HIF-1alpha), and (c) angiogenesis related phenotypes (vitronectin-directed migration, and cord formation) in HUVEC. Results: (1) O plus C in combination with V caused a regression of the in vivo growth of established tumors by 50% which was evident in both the BRCA-wt TNBC models tested. Interestingly, a marked suppression of the progression of tumor-growth was observed in the O plus C arm. (2) In vitro , V alone (10 µM) inhibited baseline as well as EGF-induced phosphorylation of AKT (S473/T308), S6RP, 4EBP1 and ERK. (3) TNBC cells exhibited higher sensitivity to V in clonogenic assays when combined with a 10 µM fixed dose of O and C/temozolomide. (4) A combination of V with O plus C increased cleaved caspase-3, PARP cleavage, and pro-apoptotic signals, while inhibiting vascular mimicry, migration, and invasion in MDA-MB231, MDA-MB468, and SUM149 cells. (5) Treatment with V blocked cord formation, migration, EGF-induced HIF-1α accumulation, and phosphorylation of AKT, 4EBP1, and ERK in HUVEC. Conclusion: A profound anti-tumor efficacy of O plus C in combination with V in BRCA-wt TNBC model can be explained by a significant anti-proliferative/pro-apoptotic and anti-migratory/anti-invasive actions of the drugs (alone or in combination), as observed both in tumor cells as well as in endothelial compartments. The combination of O plus C plus V merits further investigation in TNBC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-19-03.

Abstract 2935: Anti-proliferative and pro-apoptotic effects of Olaparib, in triple negative subset of breast cancer: Does tumor suppressor PTEN play a role

Triple-negative breast tumors (TNBT) are associated with poor prognosis and high recurrence-rates after adjuvant therapy. Currently, there is no preferred standard form of chemotherapy for TNBT. Landmark studies on DNA damage checkpoints and associated repair function in preneoplastic and neoplastic cells have focused attention on the importance of the BRCA-RAD51 repair-pathway in the development and progression of TNBC. The PARP inhibitor (PARPi), Olaparib, is currently being tested in phase I / II trials in BC and holds promise for the treatment of BRCA-deficient basal-like and /or TNBC. PTEN protein/function is downregulated in ∼ 40% of breast cancer, including TNBT, and a recurrent gross-mutation of the PTEN gene is identified in breast cancer with deficient double-strand base-repair. We hypothesize that there is a cellular threshold for error-free DNA-repair in TNBT cells and that the absence of PTEN can sensitize these cells to a concurrent treatment of DNA-damaging agent (carboplatin) and PARPi (Olaparib). We determined: (a) the time course of expression of PAR protein following Olaparib in BRCA-incompetent TNBT cell lines, (b) enzymatic activity of PARPi in BRCA-competent MDA-MB-231(PTEN+), MDA468 (PTEN-null), and BT20, TNBT cells, (c) pro-apoptotic signals in both BRCA1-incompetent and BRCA1-competent TNBT cells, (d) anti-proliferative effect of Olaparib plus carboplatin on cell- survival (MTT assay, EC50s, automated live-cell counter), and 2D clonogenic assay. We have demonstrated: (1) a time dependent decrease in PAR expression with little alteration in the expression of total PARP within 24 hours of treatment of Olaparib in HCC1937 cells, (2) a comparable pattern of decrease in the levels of PAR expression in MDA-MB-231, MDA468, and BT20 cells following Olaparib and carboplatin at 24 and 48 hours, (3) a significant increase in the expression of cleaved-caspase 3 and 9, (4) a significant increase in the expression of cleaved-PARP, a downstream product of cleaved-caspase 3, (5) an increase in the TUNNEL-positive cells in response to Olaparib plus carboplatin and (6) a significant decrease in the percentage of cell-survival following combination-treatment. Strikingly, we found that amongst different BRCA-competent TNBT cells, PTEN-null MDA-MB468 cells appeared the most sensitive to the combination of Olaparib plus carboplatin with respect to EC50s, clonogenic assay, pro-apoptotic signals, and cell survival. Herein, we report a non-redundant function of PTEN in PARPi-mediated anti-proliferative and pro-apoptotic signals in TNBC. We are currently pursuing studies a) to delineate the mechanistic relationship between the thresholds of DNA repair and PTEN and b) to understand the phosphatase-independent role of PTEN in PARPi-mediated anti-proliferative / pro-apoptotic signals, the results of which will be presented in the meeting. 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 2935. doi:10.1158/1538-7445.AM2011-2935

P3-18-02: A Combination of Pathway-Targeted Inhibitor with DNA-Repair Inhibitor: Preclinical Efficacy of Zactima and Olaparib in Triple Negative Subset of Breast Cancer.

Introduction: Pathway-targeted therapy has not been established for the treatment of Triple Negative (TN) subset of breast cancer (BC). Despite emerging data supporting the use of polyADP-ribose polymerase (PARP) inhibitors, complete and durable responses are rare in TNBC and exploration of additional pathway-targeted therapies is needed. The high frequency (72-75%) of EGFR expression in TNBCs suggests that loss of BRCA1 may be coupled, either directly or indirectly, with EGFR overexpression in breast cancer ( Van der Groep et al., 2006) , and a recent report shows that even a partial loss of BRCA1 leads to an overall increase in EGFR expression in mammary epithelial cells ( Burga et al., 2011). We hypothesize that the combined inhibition of growth factor driven EGFR pathway (by Zactima, a small molecule kinase inhibitor of EGFR and VEGFR-2) and DNA-repair pathway (by Olaparib, PARP inhibitor) in the presence of DNA-damaging agent (carboplatin) will have anti-proliferative and anti-migratory effects on triple negative breast tumor (TNBT) cells. Methods : The effects of Zactima, Olaparib (single or in combination) and carboplatin were studied on: (a) the cell survival/proliferation (MTT, SRB, & cell titer-GLO assay), (b) EGF-induced upregulation of downstream proliferation markers, (c) the cellular signals for apoptosis, (d) fibronectin-directed migration (scratch-assay), (e) the vascular mimicry, and (f) the clonogenic survival (3D-ON-TOP assay) in different TNBT cell lines. Results : Our in vitro data show that, (1) Zactima (10 uM) blocked EGF-induced phosphorylation of AKT (S473 & T308), S6RP, 4EBP1 and ERK as early as 1 hr (after the treatment), (2) the range of EC50s for Zactima (96 hrs) varied from 5 uM −15 uM (HCC70, HCC1937, MDA-MB231, MDA-MB468, BT20), (3) EGFR over-expressing, PTEN-null, and ATM kinase mutated MDA-MB468 cell line exhibited lowest EC50 for Zactima as compared to other TNBT cell lines and was found to be the most sensitive cell line to Zactima (∼ 0.05 uM) when combined with 10 uM fixed dose of Olaparib, (4) a combination of Zactima with Olaparib plus carboplatin altered an adhesion-dependent cell-survival, increased caspase3 activity (expression of cleaved caspase3 and cleaved PARP) time dependently, inhibited vascular mimicry (at 6 and 24 hrs), blocked fibronectin-directed migration (scratch assay, 24 hrs), changed the organization of actin-cytoskeleton, and suppressed clonogenic growth in different TNBT cells lines. Conclusion : Zactima alone showed anti-proliferative/pro-apoptotic, and anti-migratory effects in TNBT cells. The combination of Zactima with Olaparib plus carboplatin was most effective in blocking the clonogenic growth of TNBT cells. We are currently pursuing studies to, (1) delineate the relationship between the anti-proliferative effects (clonogenic assay) of Zactima (alone or in combination) and the status of the EGFR/PI3K pathway using PIK3CA-mutated, PTEN-null and EGFR overexpressed TNBT cell lines, and (2) determine the effect of Zactima on angiogenesis using HUVEC cells, the results of which will be presented in the meeting. This work was generously supported by AVON foundation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-18-02.