Alice Shia

Phase II Randomized Preoperative Window-of-Opportunity Study of the PI3K Inhibitor Pictilisib Plus Anastrozole Compared With Anastrozole Alone in Patients With Estrogen Receptor–Positive Breast Cancer

PURPOSE Preclinical data support a key role for the PI3K pathway in estrogen receptor-positive breast cancer and suggest that combining PI3K inhibitors with endocrine therapy may overcome resistance. This preoperative window study assessed whether adding the PI3K inhibitor pictilisib (GDC-0941) can increase the antitumor effects of anastrozole in primary breast cancer and aimed to identify the most appropriate patient population for combination therapy. PATIENTS AND METHODS In this randomized, open-label phase II trial, postmenopausal women with newly diagnosed operable estrogen receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancers were recruited. Participants were randomly allocated (2:1, favoring the combination) to 2 weeks of preoperative treatment with anastrozole 1 mg once per day (n = 26) or the combination of anastrozole 1 mg with pictilisib 260 mg once per day (n = 49). The primary end point was inhibition of tumor cell proliferation as measured by change in Ki-67 protein expression between tumor samples taken before and at the end of treatment. RESULTS There was significantly greater geometric mean Ki-67 suppression of 83.8% (one-sided 95% CI, ≥ 79.0%) for the combination and 66.0% (95% CI, ≤ 75.4%) for anastrozole (geometric mean ratio [combination:anastrozole], 0.48; 95% CI, ≤ 0.72; P = .004). PIK3CA mutations were not predictive of response to pictilisib, but there was significant interaction between response to treatment and molecular subtype (P = .03); for patients with luminal B tumors, the combination:anastrozole geometric mean ratio of Ki-67 suppression was 0.37 (95% CI, ≤ 0.67; P = .008), whereas no significant Ki-67 response was observed for pictilisib in luminal A tumors (1.01; P = .98). Multivariable analysis confirmed Ki-67 response to the combination treatment of patients with luminal B tumors irrespective of progesterone receptor status or baseline Ki-67 expression. CONCLUSION Adding pictilisib to anastrozole significantly increases suppression of tumor cell proliferation in luminal B primary breast cancer.

Identification of Endoglin as an epigenetically regulated tumour-suppressor gene in lung cancer

Background:The transforming growth factor-beta (TGF- β) pathway has been implicated in proliferation, migration and invasion of various cancers. Endoglin is a TGF-β accessory receptor that modulates signalling. We identified Endoglin as an epigenetically silenced tumour-suppressor gene in lung cancer by means of a genome-wide screening approach, then sought to characterise its effect on lung cancer progression.Methods:Methylation microarray and RNA sequencing were carried out on lung cancer cell lines. Epigenetic silencing of Endoglin was confirmed by methylation and expression analyses. An expression vector and a 20-gene expression panel were used to evaluate Endoglin function. Pyrosequencing was carried out on two independent cohorts comprising 112 and 202 NSCLC cases, respectively, and the impact of Endoglin methylation on overall survival (OS) was evaluated.Results:Methylation in the promoter region resulted in silencing of Endoglin, which could be reactivated by demethylation. Increased invasion coupled with altered EMT marker expression was observed in cell lines with an epithelial-like, but not those with a mesenchymal-like, profile when Endoglin was absent. Methylation was associated with decreased OS in stage I but not in stages II–III disease.Conclusions:We show that Endoglin is a common target of epigenetic silencing in lung cancer. We reveal a link between Endoglin silencing and EMT progression that might be associated with decreased survival in stage I disease.

Abstract P3-03-12: CDK4/6 inhibitor resistant ER-positive cells remain dependent on estrogen signalling and retain sensitivity to endocrine therapy

Background: Dysregulation of the cyclin D-CDK4/6-Rb pathway is a frequent feature of ER positive breast cancers and has been linked with endocrine resistance. Several randomised trials have shown that CDK4/6 inhibitors in combination with endocrine treatment can substantially improve the outcome of patients with metastatic ER positive breast cancer. With increased clinical use of CDK4/6 inhibitors acquired resistance is emerging as a major clinical challenge. It is critical to understand the sensitivity and responsiveness of these resistant cells to estrogen to consider continued use of endocrine therapy in combination with CDK4/6 inhibition. Results: Long-term culture of the ER positive cell lines T47D and MCF7 with increasing doses of drug generated distinct clones with acquired resistance to either palbociclib or ribociclib. ESR1 expression was stable across all resistant clones. All clones continued to be reliant on estrogen for cell growth, with estrogen deprivation inducing cell death. Resistant clones still responded to estrogen stimulation. Estradiol treatment induced at least 2-fold transcriptional upregulation of pS2 and c-myc; the induction was inhibited by tamoxifen and fulvestrant treatment. Both palbociclib and ribociclib resistant clones remained sensitive to endocrine therapy, with tamoxifen IC50 values equivalent to the parental T47D and MCF7 cell lines. Mechanisms of resistance were different between the different CDK4/6 inhibitors and within the two cell lines. Palbociclib resistant MCF7 and T47D clones all increase cyclin E protein levels, but only T47D transcriptionally upregulated cyclin E. MCF7 clones transcriptionally upregulated cyclin D, with corresponding protein increase. Therefore, CDK2/cyclin E complexes seem to drive progression in MCF7 cells, whilst CDK2/cyclin D complexes function in T47D cells. Ribociclib resistant clones presented with unchanged or even decreased CDK2 expression, but demonstrated notable increase in E2F1 , indicating the requirement of a non-canonical CDK-cyclin pairing independent of CDK2. Cell lines did not exhibit complete cross resistance to all CDK4/6 inhibitors. All ribociclib-resistant T47D and MCF7 clones conferred cross-resistance to palbociclib. The majority of clones were also resistant to abemaciclib, although one T47D clone retained abemaciclib sensitivity. All palbociclib-resistant clones were equally resistant to ribociclib. Conversely, whilst one T47D ribociclib clone exhibited marked cross resistance to abemaciclib, the majority of clones remained sensitive to treatment with abemaciclib. This highlights potential different resistant mechanisms for abameciclib independent characterised cell cycle changes for palbociclib and ribociclib. Conclusions: Cells resistant to CDK4/6 inhibitors remain dependent on estrogen signalling and retain sensitivity to endocrine therapy. We continue to show that resistance is mediated by non-canonical CDK-cyclin pairings. Palbociclib and ribociclib resistant clones confer cross-resistance to both Palbociclib and ribociclib but incomplete cross-resistance to abemaciclib. Citation Format: Lenihan C, Bouchekioua-Bouzaghou K, Abdulghani R, Chupin J, Shia A, Schmid P. CDK4/6 inhibitor resistant ER-positive cells remain dependent on estrogen signalling and retain sensitivity to endocrine therapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-12.

Epigenetic Regulation of EMT in Non-Small Cell Lung Cancer.

Lung cancer remains the most diagnosed cancer in the world, with a high mortality rate and fewer therapeutic options. The most common lung cancer is non-small cell, consisting of adenocarcinoma, squamous cell carcinoma and large cell lung carcinoma. As per all solid tumours, the changes that occur for the initiation and metastasis of lung cancer can be described using the EMT (epithelial mesenchymal transition). Cells progressing through EMT lose their epithelial cell characteristics, expressing more mesenchymal markers and are phenotypically different. The transition can be controlled by changes in various pathways, such as TGF-β, PI3K, MAPK, Hedgehog and Wnt. The changes in those pathways can be controlled epigenetically, via DNA methylation, histone modifications or changes in small/non-coding RNA. We will describe the epigenetic changes that occur in these pathways and how we can consider novel methods to generate a synthetic lethality target in an epigenetically regulated pathway in EMT.

Abstract P3-06-02: Characterization of resistance to the selective CDK4/6 inhibitor palbociclib in ER positive breast cancer

Background: Dysregulation of the cyclin D-CDK4/6-Rb axis occurs in a substantial proportion of ER-positive (ER+) breast cancers and has been linked with endocrine resistance. Adding the CDK4/6 inhibitor palbociclib to endocrine treatment has led to a substantial improvement of the outcome of patients with ER+ metastatic breast cancer. However, with the increasing clinical use, acquired resistance to palbociclib is merging as a new major clinical challenge. Methods: The ER+ cell lines T47D and MCF7 have been shown to be highly sensitive to treatment with palbociclib. Using long-term co-culture with increasing doses of Palbociclib, we generated MCF7 and T47D cell line clones with acquired resistance to Palbociclib. Three distinct resistant clones were selected for each cell line showing an IC50 shift from sensitive to resistant of approximately 300nM to 3uM for MCF7 and 400nM to 3.5uM for T47D, respectively. Resistant cell lines were characterized using RNA sequencing and mass spectrometry-based phosphoproteomics. Effects on selected target proteins (eg pAKT, pS6, pRB, RB or Cyclin D1) were confirmed using Western Blots. To modify resistance to palbociclib, a targeted in vitro drug-screen was performed using a range of inhibitors of the PI3K/AKT/mTOR and MEK pathways. Results: Western blot analysis of resistant cell lines demonstrated sustained down-regulation of Rb and phospho-Rb in response to palbociclib, which was reversible after discontinuation of palbociclib. Mass spectrometry identified >6,000 peptides across parental and resistant cells corresponding to 4,757 phospho-peptides and 5,337 phosphorylation sites. Pathway analysis suggested increased activity in the P3IK/AKT/mTOR pathway in resistant clones (including Akt1, p90S6K and mTOR), as well as changes in p53 and apoptotic regulation (e.g. phosphorylation of BAD). In addition, resistant clones showed multiple phosphorylation changes in the Rho/Rac pathway, suggesting changes in cytoskeletal organisation and a more invasive phenotype. Targeted drug screening showed a variable pattern across resistant clones with increased sensitivity to co-treatment of palbociclib with AKT inhibitors, PI3K alpha/delta inhibitors and/or MEK inhibitors in selected resistant clones, whereas pan-PI3K or PI3K beta/delta inhibitors showed limited efficacy in the selected clones. Conclusions: Phosphoproteomic analysis of palbociclib-resistant ER+ breast cancer cell lines demonstrated up-regulation of PI3K/AKT/mTOR and anti-apoptotic pathways. Resistant cell lines were sensitive to inhibition of PI3K/AKT/mTOR and/or MEK pathways with distinct patterns of activity across resistant clones suggesting that co-treatment of CDK4/6 inhibitors and PI3K/AKT and/or MEK inhibitors warrants further investigation as potential new therapeutic strategies in palbociclib resistance. Citation Format: Lenihan C, Bouchekioua-Bouzaghou K, Shia A, Wilkes E, Casado-Izquierdo1 P, Cutillas P, Schmid P. Characterization of resistance to the selective CDK4/6 inhibitor palbociclib in ER positive breast cancer. [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 P3-06-02.

Abstract 3759: Inhibition of heat shock protein 27 (Hsp27) expression by apatorsen as a therapeutic strategy in breast cancer

Background: Heat shock protein 27 (Hsp27) is over-expressed in a number of cancers, including breast cancer, and interacts with apoptotic proteins inhibiting the apoptotic process and promoting tumourgenesis. Clinically, Hsp27 expression has been associated with a more aggressive phenotype and resistance to chemotherapy. Apatorsen (OGX-427) is a therapeutic antisense oligonucleotide designed to specifically target Hsp27. Apatorsen has shown single agent clinical activity, and randomised trials in combination with chemotherapy are underway. Here we present pre-clinical validation of inhibition of Hsp27 expression by apatorsen as a novel therapeutic strategy in breast cancer. Results: Basal expression analysis of Hsp27 by qPCR and western blot in a panel of >20 breast cancer cell lines demonstrated a wide variation with up to 3-log difference in expression between cell lines and good correlation of RNA and protein expression levels. Hsp27 expression increased after treatment with chemotherapy agents. Treatment with apatorsen caused rapid reduction of cellular Hsp27 levels within 24-48 hours; effects on Hsp27 levels were sustained for 5 and 7 days. Knockdown of Hsp27 was associated with significant reduction of cell survival with 38-93% loss of cell viability across the cell line panel (compared to mismatched oligonucleotide). There was no association between response to apatorsen and basal Hsp27 expression or molecular breast cancer subtype. Using whole genome mRNA (Illumina Human HT-12 v4 Expression BeadChip Kit) and methylation analysis (Illumina Human 450K Methylation BeadChip), we were unable to define a predictive signature of response to apatorsen. Co-treatment of breast cancer cell lines with apatorsen and Doxorubicin, Paclitaxel and Vinorelbine chemotherapy showed an additive cytotoxic effect. In vivo validation of the effects of apatorsen is ongoing. Conclusions: Therapeutic silencing of Hsp27 expression in breast cancer cell lines with apatorsen presents a novel strategy for the treatment of disease. Here we present data to support the use of apatorsen in combination with conventional chemotherapy for the treatment of breast cancer. Citation Format: Emilia Komulainen, Alice Shia, Jasmin Bansal, Francessa Cavicchioli, Karen O’Leary, John Foster, Peter Schmid. Inhibition of heat shock protein 27 (Hsp27) expression by apatorsen as a therapeutic strategy in breast cancer. [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 3759.