PDF file 299K, Additional characterization of human breast cancer cell lines; immunofluorescence analysis and lung retention assays

PDF file 325K, Additional validation of ST6GalNAc2 as a metastasis suppressor including in a spontaneous metastasis assay using the parental 4T1 cells

XLSX file - 1167K, Gene Set Expression Analysis (GSEA) performed using default values for all the parameters. Also includes GDNF-RGS gene list and GO Analysis.

PDF file - 4112K, Characterization of long-term E2 deprived (LTED) breast cancer cell line (S1); Characterization of the MCF7-2A model (S2); Treatment of MCF7 cells with ICI 182,780 blocks ERdependent GDNF signaling (S3); Generation of the GDNF-RGS (S4); GDNF-RGS positivity correlates with poor outcome in human breast cancer (S5); Kaplan-Meier analyses for breast cancer cases stratified by GDNF-RGS (S6); Correlation of GDNF-RGS with Ki67 staining following anastrozole treatment (S7); ER H-score values in 52 paired primary tumor samples pre-aromatase inhibitor treatment (pre-AI) and recurrent/metastatic tumors following adjuvant AI treatment (post-AI) (S8).

PDF file 149K, In vivo validation of 3 hits identified in the screen (Mre11a, Fen1, Wwc1)

PDF file 174K, Additional characterization of the 4T1-Luc cells in lung retention assays

Supplementary Figure 3 from The Vascular Endothelial Growth Factor Receptor Inhibitor PTK787/ZK222584 Inhibits Aromatase

PDF file 107K, Inventory of supplementary material. Supplementary Table S1 listing primers and siRNAs. Legends for Supplementary Fig. S1 - S7 Legends for Supplementary Movies S1 and S2 Supplementary Figures S1 - S6 legends Supplementary Movies S1 and S2 legends

MOV file 2886K, Video showing dynamic flow adhesion assays of ZR75.1 shNTC and shST6 cells on a HUVEC monolayer in the presence of the galectin-3 inhibitor GCS-100

PDF file 60K, Characterization of the MDA-MB-231 cells stably expressing vector or mouse ST6GalNAc2

Supplementary Figure 1 from Gene and Protein Expression Profiling of Human Ovarian Cancer Cells Treated with the Heat Shock Protein 90 Inhibitor 17-Allylamino-17-Demethoxygeldanamycin

Supplementary Figure Legends 1-4, Table Legend from The Vascular Endothelial Growth Factor Receptor Inhibitor PTK787/ZK222584 Inhibits Aromatase

PDF file 95K, Data demonstrating that siRNA mediated downregulation of ST6GalNAc2 expression in human and mouse cell lines promotes tumor cell aggregation in vivo

Mechanistic target of rapamycin (mTOR) promotes cell proliferation, growth, and survival and is overactivated in many tumors and central nervous system disorders. PQR620 (3) is a novel, potent, selective, and brain penetrable inhibitor of mTORC1/2 kinase. PQR620 (3) showed excellent selectivity for mTOR over PI3K and protein kinases and efficiently prevented cancer cell growth in a 66 cancer cell line panel. In C57BL/6J and Sprague-Dawley mice, maximum concentration ( Cmax) in plasma and brain was reached after 30 min, with a half-life ( t1/2) > 5 h. In an ovarian carcinoma mouse xenograft model (OVCAR-3), daily dosing of PQR620 (3) inhibited tumor growth significantly. Moreover, PQR620 (3) attenuated epileptic seizures in a tuberous sclerosis complex (TSC) mouse model. In conclusion, PQR620 (3) inhibits mTOR kinase potently and selectively, shows antitumor effects in vitro and in vivo, and promises advantages in CNS indications due to its brain/plasma distribution ratio.

Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclinical characterization of compound 1 (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concentrations. No off-target interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, 1 did not bind tubulin, which was observed for the structurally related 4 (BKM120, buparlisib). Compound 1 is orally available, crosses the blood-brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compound 1 demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compound's safety profile, identifies 1 as a clinical candidate with a broad application range in oncology, including treatment of brain tumors or CNS metastasis. Compound 1 is currently in phase II clinical trials for advanced solid tumors and refractory lymphoma.

BKM120 (Buparlisib) is one of the most advanced phosphoinositide 3-kinase (PI3K) inhibitors for the treatment of cancer, but it interferes as an off-target effect with microtubule polymerization. Here, we developed two chemical derivatives that differ from BKM120 by only one atom. We show that these minute changes separate the dual activity of BKM120 into discrete PI3K and tubulin inhibitors. Analysis of the compounds cellular growth arrest phenotypes and microtubule dynamics suggest that the antiproliferative activity of BKM120 is mainly due to microtubule-dependent cytotoxicity rather than through inhibition of PI3K. Crystal structures of BKM120 and derivatives in complex with tubulin and PI3K provide insights into the selective mode of action of this class of drugs. Our results raise concerns over BKM120's generally accepted mode of action, and provide a unique mechanistic basis for next-generation PI3K inhibitors with improved safety profiles and flexibility for use in combination therapies.

BACKGROUND:Therapies targeting estrogenic stimulation in estrogen receptor-positive (ER+) breast cancer (BC) reduce mortality, but resistance remains a major clinical problem. Molecular studies have shown few high-frequency mutations to be associated with endocrine resistance. In contrast, expression profiling of primary ER+ BC samples has identified several promising signatures/networks for targeting.METHODS:To identify common adaptive mechanisms associated with resistance to aromatase inhibitors (AIs), we assessed changes in global gene expression during adaptation to long-term estrogen deprivation (LTED) in a panel of ER+ BC cell lines cultured in 2D on plastic (MCF7, T47D, HCC1428, SUM44 and ZR75.1) or in 3D on collagen (MCF7) to model the stromal compartment. Furthermore, dimethyl labelling followed by LC-MS/MS was used to assess global changes in protein abundance. The role of target genes/proteins on proliferation, ER-mediated transcription and recruitment of ER to target gene promoters was analysed.RESULTS:The cholesterol biosynthesis pathway was the common upregulated pathway in the ER+ LTED but not the ER- LTED cell lines, suggesting a potential mechanism dependent on continued ER expression. Targeting the individual genes of the cholesterol biosynthesis pathway with siRNAs caused a 30-50 % drop in proliferation. Further analysis showed increased expression of 25-hydroxycholesterol (HC) in the MCF7 LTED cells. Exogenous 25-HC or 27-HC increased ER-mediated transcription and expression of the endogenous estrogen-regulated gene TFF1 in ER+ LTED cells but not in the ER- LTED cells. Additionally, recruitment of the ER and CREB-binding protein (CBP) to the TFF1 and GREB1 promoters was increased upon treatment with 25-HC and 27-HC. In-silico analysis of two independent studies of primary ER+ BC patients treated with neoadjuvant AIs showed that increased expression of MSMO1, EBP, LBR and SQLE enzymes, required for cholesterol synthesis and increased in our in-vitro models, was significantly associated with poor response to endocrine therapy.CONCLUSION:Taken together, these data provide support for the role of cholesterol biosynthesis enzymes and the cholesterol metabolites, 25-HC and 27-HC, in a novel mechanism of resistance to endocrine therapy in ER+ BC that has potential as a therapeutic target.

Over 80% of breast cancers (BC) at primary diagnosis express the estrogen receptor (ER+). Therapies targeting the estrogenic stimulation of tumor growth reduce mortality from ER+ BC but resistance remains a major clinical problem. Data from large studies such as TCGA indicate that other than a small number of high frequency mutations such as TP53, PIK3CA and GATA3 that have little association with endocrine resistance, primary ER+ BC shows very low frequency of individual mutations making targeting difficult. In contrast, expression profiling of primary ER+ BC samples have identified several promising signatures/networks for targeting. In order to identify common adaptive mechanisms associated with resistance to aromatase inhibitors, we assessed changes in global gene expression during adaptation to long-term estrogen deprivation (LTED) in a panel of ER+ BC cell lines cultured in 2D on plastic (MCF7, T47D, HCC1428, SUM44, MDA-MB-361 and ZR75.1) or 3D on collagen to model the stromal compartment (MCF7). At the point of resistance MCF7-LTED, HCC1428-LTED and SUM44-LTED retained ER expression whilst T47D-LTED, ZR75.1-LTED and MDA-MB-361-LTED reduced or lost ER. In order to identify common adaptive mechanisms, ingenuity pathway was used. Genes submitted for analysis were selected on the basis of a p-value of 0.001, FDR 5% and fold change 1.5 relative to the parental cell lines. Strikingly, the cholesterol biosynthesis pathway was the common up-regulated pathway in the ER+ LTED cell lines but not in the ER- LTED cell lines, suggesting a potential mechanism dependent on continued ER expression. Further interrogation showed increased expression of MSMO1 (x2.27), EBP (x1.57), SQLE (x2.37) and IDI1 (x2.74) in the MCF7-LTED. As mRNA does not always correlate with protein, we applied quantitative proteomics in wt-MCF7 and MCF7-LTED cells using dimethyl labelling followed by LC-MS/MS. Several enzymes within the cholesterol biosynthesis pathway were higher in abundance in the MCF7-LTED such as EBP (x1.54), SQLE (x3.24) and LBR (x1.86). Evidence suggests that patients with cancer accumulate cholesteryl esters as a result of loss of PTEN or PI3K/AKT/mTOR activation, stimulating SREBP, ACAT1, HMGSCR and LDLR. As 40% of ER+ BC harbor PTEN loss or PIK3CA mutations, we assessed levels of SREBP, HMGCR, LDLR or ACAT1 in our data sets. Neither SREBP nor HMGCR mRNA or protein levels changed in the ER+ LTED or ER- LTED. Furthermore, ACAT1 was downregulated in the ER+ LTED. Moreover, levels of free and esterified cholesterol in wt-MCF7 and MCF7-LTED cell lysates were unchanged. Previous studies have suggested oxysterols can act as selective ER-modulators (SERM). We subsequently investigated the function of these cholesterol metabolites in our LTED models. Treatment with 25-hydroxycholesterol (HC) or 27-HC increased ER mediated-transcription and expression of the endogenous estrogen-regulated gene TFF1 in ER+ LTED cells but not in the ER- LTED. Targeting the individual genes of the cholesterol biosynthesis pathway with siRNAs caused a c. 30-60% drop in proliferation and concomitant drop in TFF1 expression in the ER+ LTED cells together with a reduction in recruitment of the ER and CREB binding protein (CBP) to the TFF1 promoter. Gene expression data from 704 primary ER+ BC from patients treated with adjuvant tamoxifen showed that MSMO1 (p=0.047), EBP (p=0.043), SQLE (p=0.0001), DHCR7 (p=0.002) and IDI1 (p=0.0005), enzymes required for cholesterol synthesis and up-regulated in our in-vitro models, were associated with poor relapse free survival. Of note, DHCR7 forms part of the EndoPredict gene signature. Taken together, these data provide support for the role of the cholesterol biosynthesis pathway and its metabolites as a novel mechanism of resistance to endocrine therapy in ER+ BC and its potential as a therapeutic target. Citation Format: Nikiana Simigdala, Qiong Gao, Sunil Pancholi, Marketa Zvelebil, Ricardo Ribas, Mitch Dowsett, Lesley-Ann Martin. Cholesterol biosynthesis pathway as a novel mechanism of resistance identified in ER+ long-term estrogen deprived cells. [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 A37.

Abstract Due to its central role in growth, proliferation, survival and migration, phosphoinositide 3-kinase (PI3K) is considered as an important drug target in oncology (1). BKM120 is one of the clinically most advanced PI3K inhibitors (PI3Ki), and is currently listed in more than 80 clinical studies aimed at attenuating tumour progression. As an off-target effect, BKM120 was reported to disrupt microtubules (MT) at concentrations around 1 μM (2). Here, we elucidate in detail the structural factors defining PI3K- and tubulin-binding of BKM120, and present a pure PI3K inhibitor (PQR309) and a potent MT disruptor (MTD147) differing from BKM120 by only 1 Dalton. Separation of PI3Ki and MT disruption activities of BKM120 allowed profiling of BKM120 against PQR309 and MTD147: cellular growth profiles of PQR309 clustered with other PI3Ki such as GDC0941/GDC0980, while BKM120 matched MTD147. Both yielded a G2/M cell cycle arrest with typical histone3 phosphorylation. Accumulation of G2/M arrested cells was already evident at concentrations yielding 50% growth inhibition. Interestingly, BKM120 concentrations for 50% cell growth inhibition (with evident G2/M arrest) ranged below or within its reported AUC0-24 levels at day 8 in patient plasma (3,4). This result implies that the two activities of BKM120 cannot be separated, thus complicating the understanding of drug action and impacting on the rational of combination therapies at relevant drug doses. Using X-ray crystallography we found that BKM120 binds to the colchicine pocket on β-tubulin. This study further highlights the importance of the pyrimidine core orientation for tight tubulin binding. Interestingly, activities of regio-isomers of the pyrimidine core are inversed for PI3Ki and tubulin association, and modulate binding by a factor of >30x. Finally, a combination of biochemical, cellular and structural data suggests an inverted orientation of BKM120 in the catalytic cleft of PI3K as previously proposed (6). In summary, the dissection of BKM120 functions allows reassessment of its dominant activity, to increase drug safety, and to flexibly control PI3K and/or MT targeting in combination therapy. 1. M. P. Wymann, R. Schneiter, Nat Rev Mol Cell Biol 9, 162 (2008).; 2. S. M. Brachmann et al., Mol Cancer Ther 11, 1747 (2012). 3. J. C. Bendell et al., J Clin Oncol 30, 282 (2012). 4. C. Saura et al., Clin Cancer Res 20, 1935 (2014). 5. A. E. Prota et al., Science 339, 587 (2013). 6. S. M. Maira et al., Mol Cancer Ther 11, 317 (2012). Citation Format: Thomas Bohnacker, Florent Beaufils, Andrea E. Prota, John E. Burke, Anna Melone, Alison J. Inglis, Ludovico Fusco, Vladimir Cmiljanovic, Natasa Cmiljanovic, Denise Rageot, Katja Bargsten, Gonzalo Saez-Calvo, Olivier Pertz, Amol B. Aher, Anna Akhmanova, Fernando J. Diaz, Doriano Fabbro, Marketa Zvelebil, Roger L. Williams, Michel O. Steinmetz, Matthias P. Wymann. BKM120-mediated G2 arrest: Structural and functional segregation of off-target action and PI3K inhibition. [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 671. doi:10.1158/1538-7445.AM2015-671

INTRODUCTION:Triple-negative breast cancer (TNBC) is a heterogeneous group of tumours in which chemotherapy, the current mainstay of systemic treatment, is often initially beneficial but with a high risk of relapse and metastasis. There is currently no means of predicting which TNBC will relapse. We tested the hypothesis that the biological properties of normal stem cells are re-activated in tumour metastasis and that, therefore, the activation of normal mammary stem cell-associated gene sets in primary TNBC would be highly prognostic for relapse and metastasis.METHODS:Mammary basal stem and myoepithelial cells were isolated by flow cytometry and tested in low-dose transplant assays. Gene expression microarrays were used to establish expression profiles of the stem and myoepithelial populations; these were compared to each other and to our previously established mammary epithelial gene expression profiles. Stem cell genes were classified by Gene Ontology (GO) analysis and the expression of a subset analysed in the stem cell population at single cell resolution. Activation of stem cell genes was interrogated across different breast cancer cohorts and within specific subtypes and tested for clinical prognostic power.RESULTS:A set of 323 genes was identified that was expressed significantly more highly in the purified basal stem cells compared to all other cells of the mammary epithelium. A total of 109 out of 323 genes had been associated with stem cell features in at least one other study in addition to our own, providing further support for their involvement in the biology of this cell type. GO analysis demonstrated an enrichment of these genes for an association with cell migration, cytoskeletal regulation and tissue morphogenesis, consistent with a role in invasion and metastasis. Single cell resolution analysis showed that individual cells co-expressed both epithelial- and mesenchymal-associated genes/proteins. Most strikingly, we demonstrated that strong activity of this stem cell gene set in TNBCs identified those tumours most likely to rapidly progress to metastasis.CONCLUSIONS:Our findings support the hypothesis that the biological properties of normal stem cells are drivers of metastasis and that these properties can be used to stratify patients with a highly heterogeneous disease such as TNBC.