BT474 Xenografts Research Articles

Activated estrogen receptor-mitogen-activated protein kinases cross talk confer acquired resistance to lapatinib.

BackgroundThe efficacy of lapatinib is limited by the development of acquired resistance. The aim of this study was to investigate the role of estrogen receptor (ER) signaling compensatory activation in acquired resistance to lapatinib in breast cancer cells BT474 and the related mechanism.MethodsAcquired resistant cell model resistant (r)BT474 was generated with an increasing concentration of lapatinib. Real-time polymerase chain reaction and Western blotting were used to determine the changes of human epidermal growth factor receptor (HER)2 and ER pathways in breast cancer cell BT474 after treatment with lapatinib and the distinction between BT474 and rBT474. Methyl thiazolyl tetrazolium and colony formation assays were employed to detect the proliferation of rBT474 and BT474 cells treated with lapatinib and/or an ER inhibitor, fulvestrant, respectively.ResultsLapatinib could inhibit phosphorylation of HER2 and induce expression of forkhead-box protein O3a and progesterone receptor. Acquired resistant cell model rBT474 could grow in the presence of 5 μM lapatinib, with an apoptosis rate of only 5%. Significant inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) pathway and the activation of the mitogen-activated protein kinases (MAPK) and ER pathways were detected in rBT474, compared with BT474. Furthermore, the expressions of Src phosphorylation and caveolin-1 were also upregulated. The viability of rBT474 was markedly suppressed by the lapatinib/fulvestrant combination in vitro, confirmed by the BT474 xenograft model.ConclusionER signaling compensatory activation may partly contribute to lapatinib acquired resistance in HER2-overexpressing/ERα-positive breast cancer cells, which might be related to PI3K/AKT inhibition and MAPK pathway activation.

ANG4043, a novel brain-penetrant peptide-mAb conjugate, is efficacious against HER2-positive intracranial tumors in mice.

Anti-HER2 monoclonal antibodies (mAb) have been shown to reduce tumor size and increase survival in patients with breast cancer, but they are ineffective against brain metastases due to poor brain penetration. In previous studies, we identified a peptide, known as Angiopep-2 (An2), which crosses the blood-brain barrier (BBB) efficiently via receptor-mediated transcytosis, and, when conjugated, endows small molecules and peptides with this property. Extending this strategy to higher molecular weight biologics, we now demonstrate that a conjugate between An2 and an anti-HER2 mAb results in a new chemical entity, ANG4043, which retains in vitro binding affinity for the HER2 receptor and antiproliferative potency against HER2-positive BT-474 breast ductal carcinoma cells. Unlike the native mAb, ANG4043 binds LRP1 clusters and is taken up by LRP1-expressing cells. Measuring brain exposure after intracarotid delivery, we demonstrate that the new An2-mAb conjugate penetrates the BBB with a rate of brain entry (Kin) of 1.6 × 10(-3) mL/g/s. Finally, in mice with intracranially implanted BT-474 xenografts, systemically administered ANG4043 increases survival. Overall, this study demonstrates that the incorporation of An2 to the anti-HER2 mAb confers properties of increased uptake in brain endothelial cells as well as BBB permeability. These characteristics of ANG4043 result in higher exposure levels in BT-474 brain tumors and prolonged survival following systemic treatment. Moreover, the data further validate the An2-drug conjugation strategy as a way to create brain-penetrant biologics for neuro-oncology and other CNS indications.

Abstract LB-212: HER2-overexpressing breast cancer xenografts amplify FGFR signaling upon acquisition of resistance to dual blockade with trastuzumab and lapatinib

Abstract We sought to discover mechanisms of acquired resistance to the combination of the HER2 inhibitors trastuzumab and lapatinib in HER2 gene amplified BT474 xenografts. The combination of lapatinib and trastuzumab completely eliminated BT474 tumors established in nude mice after <3 weeks of treatment. However, some tumors recurred as early as 9 weeks after the combination was stopped. Three recurrent tumors did not respond to retreatment with the combination of lapatinib and trastuzumab. More than 90% of tumors derived from these recurrent tumors did not respond to lapatinib/trastuzumab or the combination of trastuzumab and pertuzumab. Both the parental drug-sensitive xenografts and the lapatinib/trastuzumab-resistant (LTR) tumors exhibited 5 to 6-fold HER2 gene amplification as measured by FISH using probes for HER2 and chromosome 17. The LTR cancers maintained levels of P-HER2, P-AKT and P-ERK in the presence of lapatinib and trastuzumab as measured by IHC of tumor sections whereas treatment of parental BT474 xenografts reduced P-HER2 levels. LTR tumors exhibited markedly reduced in situ levels of lapatinib compared to BT474 sensitive tumors (2.157 µg lapatinib/gm tissue versus 4.701 µg lapatinib/gm tissue, p=0.02) as assessed by liquid chromatography-mass spectrometry (LC-MS). Quantitative site-specific mass spectrometric analysis of tyrosine phosphorylation from tumor lysates revealed enriched P-Y877 HER2 in LTR compared to parental BT474 xenografts. We next performed next generation sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes. There were no mutations in HER2 but we observed amplification of FGF3, FGF4, and FGF19 genes in LTR xenografts. Consistent with increased autocrine activation of FGFR, we observed markedly elevated levels of P-FGFR in LTR xenografts compared to parental xenografts as assessed by IHC. We speculate that ligand-induced FGFR signaling may induce resistance to dual HER2 blockade by 1) a tumor cell autonomous mechanism resulting in transactivation of HER2 and, 2) an alteration in the tumor microenvironment that reduces lapatinib uptake and/or retention by the LTR tumors. In conclusion, we show amplification of FGFR signaling upon acquired resistance to combinations of HER2-targeted agents. Studies with FGFR tyrosine kinase inhibitors to reverse drug resistance are in progress. Citation Format: Joan T. Garrett, Jennifer Becker, Mónica Valeria Estrada, Carlos L. Arteaga. HER2-overexpressing breast cancer xenografts amplify FGFR signaling upon acquisition of resistance to dual blockade with trastuzumab and lapatinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-212. doi:10.1158/1538-7445.AM2014-LB-212

Abstract 2988: Microenvironmental distribution of trastuzumab is heterogeneous and decreases sharply when administered following a single dose of bevacizumab in Her2+ve xenografts and metastases models

Abstract We have previously shown that trastuzumab distributes heterogeneously within MDA-435-Her2 xenografts. Though peak accumulation occurs 24h after administration & trastuzumab was detected greater than 150 µm from many vessels, there remained substantial Her2+ve areas that were negative for bound drug. Methods: Her2+ve human carcinoma xenografts were grown in NOD/SCID mice as solid tumors or as metastatic models following ip or iv injection. Tumors were MR-imaged (T1-weighted) with a 500kDa Gd-labeled hyperbranched polyglycerol (HPG) contrast agent (CA). Treatments included bevacizumab (BvMAb, 10 mg/kg), trastuzumab (TsMab, 10 mg/kg) and human IgG (10 mg/kg). Multiplexed staining and imaging of whole tumor sections was used to evaluate the distribution of TsMAb in the context of the tumor microenvironment. Findings: BT474, MDA361 and SKOV3 primary tumor xenografts all show highly heterogeneous distribution of TsMAb at 24h, where some areas are highly saturated with TsMAb and others contain unbound Her2+ tissue. Small metastases in the peritoneal cavity and lung also exhibit substantial proportions of tissue that are negative for TsMAb. CD31-stained vessels dual-labeled for a perfusion marker are often seen to have little or no bound TsMAb on perivascular tumor cells. Pretreatment with BvMAb for 24-72h results in significant reductions in tumor accumulation of TsMAb, with tumors showing 60-90% of bound TsMAb relative to IgG-treated controls (p<0.05). BvMAb -treated xenografts had reduced fractions of perfused vessels and reduced measures of vascular function assessed using DCE-MRI (49% reduction in CA, p<0.05). Corresponding parameter maps of MR-imaged high vascular function and histological images of bound TsMAb do not consistently correlate. Detailed maps of the tumor microenvironment show that even with VEGF blockade, the distribution of TsMAb still does not correspond to vessels that are perfused. Significant numbers of perfused vessels persist with no perivascular bound TsMAb despite the presence of Her2+ cells; saturation with bound TsMAb is also not consistently correlated with the presence or absence of smooth muscle, collagen or ZO-1 stained tight junctions, examined using immunohistochemistry. Summary: The extravascular distribution of TsMAb in Her2-overexpressing xenografts, including micrometastases, is heterogeneous and is not explained simply by the presence or absence of vascular function. Pre-treatment with BvMAb for even short exposures of 24-72h worsens the distribution of TsMAb within the tumor microenvironment. The persistence of tissues that are unbound for trastuzumab suggests the possibility of inadequate drug exposure as a mechanism for drug resistance that may be exacerbated by pre-treatment with a VEGF blockade. Citation Format: Jennifer H. E. Baker, Alastair H. Kyle, Stefan A. Reinsberg, Firas Moosvi, Jordan Cran, Urs Hafeli, Katayoun Saatchi, Andrew I. Minchinton. Microenvironmental distribution of trastuzumab is heterogeneous and decreases sharply when administered following a single dose of bevacizumab in Her2+ve xenografts and metastases models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2988. doi:10.1158/1538-7445.AM2014-2988

Abstract 2064: Mass Spectrometry Imaging of therapeutic antibodies: Distribution of unlabeled trastuzumab in CB.17 SCID mice implanted with the human breast BT474 xenograft

Abstract Introduction Mass Spectrometry Imaging (MSI) is an emerging technique in preclinical analysis that allows for analysis of the distribution and the quantification of target molecules in tissue sections. The main advantage of this imaging technology is the detection of the molecule of interest in tissues without labelling and with high specificity. MSI is now routinely used for small molecule distribution analysis in lead optimization process, and in PK/PD or toxicity studies. Due to the mass range limitation of the instrument analyzer one of the main challenges is the detection of administered large molecules such as therapeutic antibodies. . In this study, we describe the detection and the distribution of trastuzumab, a monoclonal antibody that recognizes the HER2/neu receptor, in a BT474 human breast xenograft model. Experimental procedures BT474 tumor fragments are implanted in female CB.17 SCID mice. When the tumors reach 80-120 mm3, the trastuzumab treatment of 20mg/kg with eight bi-weekly intraperitoneal injections was initiated. The endpoint was fixed at day 30 post first administration. At 50 hours post 1st, 5th and 8th doses vehicle and trastuzumab treated animals were sacrificed and tumor, plasma and carcass were collected. One section of each tumor tissue was first digested with a proteolytic enzyme deposited with a dedicated device onto the tissue. After the proteolytic digestion, the selected MALDI matrix is sprayed onto the tissue and then the distribution of the Trastuzumab is determined based on its specific peptide mass fingerprint (PMF) by high resolution mass spectrometry imaging with a MALDI-FTICR instrument (SolariX FTICR 7.0T from Bruker). The images are obtained with Quantinetix software dedicated to the MSI data set. Novel Aspect We describe a process which combines MSI and bottom-up proteomics approach directly on tissue to follow the distribution of a therapeutic antibody without any labelling. Until now, mass spectrometry imaging was used to study some biomarkers' (proteins) distribution on tissue. This work describes a novel MSI application for analysis of a dosed therapeutic antibody. Data collection is ongoing. Conclusions We have adapted the use of Mass Spectrometry Imaging to follow the distribution of a non-labeled therapeutic antibody based on its peptide mass fingerprint in dosed tissues. This opens the use of this technology to other large molecules, such as therapeutic proteins or Antibody-Drug Conjugates (ADCs) in a variety of therapeutic fields (Oncology, Immunology, CNS, etc.) In addition to the distribution of the target therapeutic antibody, Mass Spectrometry Imaging could also allow the detection of other important endogenous molecules in the same or adjacent tissue section, such as specific disease markers, thus allowing further understanding of PK/PD relationships. Citation Format: David Bonnel, Chassidy Hall, Robert J. Mullin, Kathryn A. Simon, Jonathan Stauber. Mass Spectrometry Imaging of therapeutic antibodies: Distribution of unlabeled trastuzumab in CB.17 SCID mice implanted with the human breast BT474 xenograft. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2064. doi:10.1158/1538-7445.AM2014-2064

Abstract 1821: HER3 activation by MET contributes to trastuzumab resistance in HER2-positive breast cancer

Abstract Background: Trastuzumab is a standard therapy for HER2-positive breast cancer patients. Majority of the patients treated with trastuzumab in metastatic disease experience relapse. MET has been reported as one possible candidate of trastuzumab resistance however, detailed mechanism is unclear. Methods: Using panel of HER2-positive cell lines, pHER3 level was assessed with trastuzumab treatment by western blot. Co-immunoprecipitation and siRNA knockdown were used to assess the interaction between HER3 and MET. Results: Phospho-HER3 as well as pAKT levels were decreased after trastuzumab treatment in trastuzumab sensitive cell lines, while there were no deactivation in the resistant cell lines. In the primary trastuzumab resistant cell lines, 5 of 6 cells expressed MET while only one of 4 cells expressed MET in the sensitive cell lines. BT474 cells were found to have increased MET level when the cells acquired resistance to trastuzumab. HCC1954 cells, MET overexpressing and primary trastuzumab cell line, were sensitized to trastuzumab when MET was knocked down. MET knockdown led to a decrease of phosphor-HER3 level in HCC1954, BT474, and BT474 trastuzumab resistant (BT-TR) cells. The paraffin-embedded tumor samples from BT474 xenograft treated with trastuzumab and HER2-positive breast cancer patients will be stained for MET expression to assess its prognostic and predictive values sin HER2 positive breast cancer. Conclusion: MET may play a key role in maintaining HER3 phosphorylation during trastuzumab treatment and resistance in HER2 positive breast cancer. Citation Format: Kenji Hashimoto, Valentine Macaulay, Anthony Kong. HER3 activation by MET contributes to trastuzumab resistance in HER2-positive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1821. doi:10.1158/1538-7445.AM2014-1821

Poly (ADP-ribose) polymerase inhibition enhances trastuzumab antitumour activity in HER2 overexpressing breast cancer

AimPoly (ADP-ribose) polymerase (PARP) inhibitors have shown promising results in Breast Cancer (BRCA) deficient breast cancer, but not in molecularly unselected patient populations. Two lines of research in this field are needed: the identification of novel subsets of patients that could potentially benefit from PARP inhibitors and the discovery of suitable targeted therapies for combination strategies. MethodsWe tested PARP inhibition, alone or combined with the anti-HER2 antibody trastuzumab on HER2+ breast cancer. We used two PARP inhibitors in clinical development, olaparib and rucaparib, as well as genetic downmodulation of PARP-1 for in vitro studies. DNA damage was studied by the formation of γH2AX foci and comet assay. Finally, the in vivo anti-tumour effect of olaparib and trastuzumab was examined in nude mice subcutaneously implanted with BT474 cells. ResultsIn a panel of four HER2 overexpressing breast cancer cell lines, both olaparib and rucaparib significantly decreased cell growth and enhanced anti-tumour effects of trastuzumab. Cells exposed to olaparib and trastuzumab had greater DNA damage than cells exposed to each agent alone. Mechanistic exploratory assays showed that trastuzumab downmodulated the homologous recombination protein proliferating cell nuclear antigen (PCNA). Combination treatment in the BT474 xenograft model resulted in enhanced growth inhibition, reduced tumour cell proliferation, and increased DNA damage and apoptosis. ConclusionTaken together, our results show that PARP inhibition has antitumour effects and increases trastuzumab activity in HER2 overexpressing breast cancer. These findings make this novel combination a promising strategy for clinical development.

The effect of mini-PEG-based spacer length on binding and pharmacokinetic properties of a 68Ga-labeled NOTA-conjugated antagonistic analog of bombesin.

The overexpression of gastrin-releasing peptide receptor (GRPR) in cancer can be used for peptide-receptor mediated radionuclide imaging and therapy. We have previously shown that an antagonist analog of bombesin RM26 conjugated to 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) via a diethyleneglycol (PEG2) spacer (NOTA-PEG2-RM26) and labeled with 68Ga can be used for imaging of GRPR-expressing tumors. In this study, we evaluated if a variation of mini-PEG spacer length can be used for optimization of targeting properties of the NOTA-conjugated RM26. A series of analogs with different PEG-length (n = 2, 3, 4, 6) was synthesized, radiolabeled and evaluated in vitro and in vivo. The IC50 values of natGa-NOTA-PEGn-RM26 (n = 2, 3, 4, 6) were 3.1 ± 0.2, 3.9 ± 0.3, 5.4 ± 0.4 and 5.8 ± 0.3 nM, respectively. In normal mice all conjugates demonstrated similar biodistribution pattern, however 68Ga-NOTA-PEG3-RM26 showed lower liver uptake. Biodistribution of 68Ga-NOTA-PEG3-RM26 was evaluated in nude mice bearing PC-3 (prostate cancer) and BT-474 (breast cancer) xenografts. High uptake in tumors (4.6 ± 0.6%ID/g and 2.8 ± 0.4%ID/g for PC-3 and BT-474 xenografts, respectively) and high tumor-to-background ratios (tumor/blood of 44 ± 12 and 42 ± 5 for PC-3 and BT-474 xenografts, respectively) were found already at 2 h p.i. of 68Ga-NOTA-PEG3-RM26. Results of this study suggest that variation in the length of the PEG spacer can be used for optimization of targeting properties of peptide-chelator conjugates. However, the influence of the mini-PEG length on biodistribution is minor when di-, tri-, tetra- and hexaethylene glycol are compared.

Therapeutic targeting of integrin αvβ6 in breast cancer.

BackgroundIntegrin αvβ6 promotes migration, invasion, and survival of cancer cells; however, the relevance and role of αvβ6 has yet to be elucidated in breast cancer.MethodsProtein expression of integrin subunit beta6 (β6) was measured in breast cancers by immunohistochemistry (n > 2000) and ITGB6 mRNA expression measured in the Molecular Taxonomy of Breast Cancer International Consortium dataset. Overall survival was assessed using Kaplan Meier curves, and bioinformatics statistical analyses were performed (Cox proportional hazards model, Wald test, and Chi-square test of association). Using antibody (264RAD) blockade and siRNA knockdown of β6 in breast cell lines, the role of αvβ6 in Human Epidermal Growth Factor Receptor 2 (HER2) biology (expression, proliferation, invasion, growth in vivo) was assessed by flow cytometry, MTT, Transwell invasion, proximity ligation assay, and xenografts (n ≥ 3), respectively. A student’s t-test was used for two variables; three-plus variables used one-way analysis of variance with Bonferroni’s Multiple Comparison Test. Xenograft growth was analyzed using linear mixed model analysis, followed by Wald testing and survival, analyzed using the Log-Rank test. All statistical tests were two sided.ResultsHigh expression of either the mRNA or protein for the integrin subunit β6 was associated with very poor survival (HR = 1.60, 95% CI = 1.19 to 2.15, P = .002) and increased metastases to distant sites. Co-expression of β6 and HER2 was associated with worse prognosis (HR = 1.97, 95% CI = 1.16 to 3.35, P = .01). Monotherapy with 264RAD or trastuzumab slowed growth of MCF-7/HER2-18 and BT-474 xenografts similarly (P < .001), but combining 264RAD with trastuzumab effectively stopped tumor growth, even in trastuzumab-resistant MCF-7/HER2-18 xenografts.ConclusionsTargeting αvβ6 with 264RAD alone or in combination with trastuzumab may provide a novel therapy for treating high-risk and trastuzumab-resistant breast cancer patients.

Lapatinib-incorporated lipoprotein-like nanoparticles: preparation and a proposed breast cancer-targeting mechanism.

Lapatinib is a dual inhibitor of EGFR and human epidermal growth factor receptor 2 (HER2), and used to treat advanced breast cancer. To overcome its poor water solubility, we constructed lapatinib-incorporated lipoprotein-like nanoparticles (LTNPs), and evaluated the particle characteristics and possible anti-breast cancer mechanisms. LTNPs (lapatinib bound to albumin as a core, and egg yolk lecithin forming a lipid corona) were prepared. The particle characteristics were investigated using transmission electron microscopy (TEM) and atomic force microscopy (AFM). The uptake and subcellular localization of LTNPs, as well as the effects of LTNPs on cell cycle were examined in BT-474 human breast cancer cells in vitro. Mice bearing BT-474 subcutaneous xenograft were intravenously injected with coumarin-6 loaded LTNPs (30 mg/kg) to study the targeting mechanisms in vivo. The LTNPs particles were generally spherical but flexible under TEM and AFM, and approximately 62.1 nm in size with a zeta potential of 22.80 mV. In BT-474 cells, uptake of LTNPs was mediated by endosomes through energy-dependent endocytosis involving clathrin-dependent pinocytosis and macropinocytosis, and they could effectively escape from endosomes to the cytoplasm. Treatment of BT-474 cells with LTNPs (20 μg/mL) induced a significant cell arrest at G0/G1 phase compared with the same concentration of lapatinib suspension. In mice bearing BT-474 xenograft, intravenously injected LTNPs was found to target and accumulate in tumors, and colocalized with HER2 and SPRAC (secreted protein, acidic and rich in cysteine). LTNPs can be taken up into breast cancer cells through specific pathways in vitro, and targeted to breast cancer xenograft in vivo via enhanced permeability and retention effect and SPARC.

Nuclear HER4 mediates acquired resistance to trastuzumab and is associated with poor outcome in HER2 positive breast cancer.

The role of HER4 in breast cancer is controversial and its role in relation to trastuzumab resistance remains unclear. We showed that trastuzumab treatment and its acquired resistance induced HER4 upregulation, cleavage and nuclear translocation. However, knockdown of HER4 by specific siRNAs increased trastuzumab sensitivity and reversed its resistance in HER2 positive breast cancer cells. Preventing HER4 cleavage by a γ-secretase inhibitor and inhibiting HER4 tyrosine kinase activity by neratinib decreased trastuzumab-induced HER4 nuclear translocation and enhanced trastuzumab response. There was also increased nuclear HER4 staining in the tumours from BT474 xenograft mice and human patients treated with trastuzumab. Furthermore, nuclear HER4 predicted poor clinical response to trastuzumab monotherapy in patients undergoing a window study and was shown to be an independent poor prognostic factor in HER2 positive breast cancer. Our data suggest that HER4 plays a key role in relation to trastuzumab resistance in HER2 positive breast cancer. Therefore, our study provides novel findings that HER4 activation, cleavage and nuclear translocation influence trastuzumab sensitivity and resistance in HER2 positive breast cancer. Nuclear HER4 could be a potential prognostic and predictive biomarker and understanding the role of HER4 may provide strategies to overcome trastuzumab resistance in HER2 positive breast cancer.

ATP directed agent, 8-chloro-adenosine, induces AMP activated protein kinase activity, leading to autophagic cell death in breast cancer cells

Background8-chloro-adenosine (8-Cl-Ado) is a unique ribonucleoside analog which is currently in a phase I clinical trial for hematological malignancies. Previously, we demonstrated in breast cancer cells that a 3-day treatment with 10 μM 8-Cl-Ado causes a 90% loss of clonogenic survival. In contrast, there was only a modest induction of apoptosis under these conditions, suggesting an alternative mechanism for the tumoricidal activity of 8-Cl-Ado.MethodsCellular metabolism, AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) pathway signaling, as well as autophagy induction was evaluated in breast cancer cell lines treated with 8-Cl-Ado. The effects of knocking down essential autophagy factors with small interfering RNA on 8-Cl-Ado-inhibited cell survival was assessed in breast cancer cells by examining apoptosis induction and clonogenic survival. In vivo efficacy of 8-Cl-Ado was measured in two breast cancer orthotopic model systems.ResultsWe demonstrate that in breast cancer cell lines, the metabolism of 8-Cl-Ado results in depletion of endogenous ATP that subsequently induces the phosphorylation and activation of the energy sensor, AMPK. This was associated with an attenuation of mTOR signaling and an induction of the phosphorylation of the autophagy factor, Unc51-like kinase 1 on Ser555. 8-Cl-Ado-mediated induction of autophagy was evident by increased aggregates of microtubule-associated protein 1 light chain 3B (LC3B) which was associated with its conversion to its lipidated form, LC3B-II, p62 degradative flux, and increased formation of acidic vesicular organelles. Additionally, transfection of MCF-7 cells with siRNA to ATG7 or beclin 1 provided partial protection of the cells to 8-Cl-Ado cytotoxicity as measured by clonogenicity. In vivo, 8-Cl-Ado inhibited growth of both MCF-7 and BT-474 xenograft tumors. Moreover, in 9 of 22 BT-474 tumors treated with 100 mg/kg/day 3 times a week, there was an absence of macroscopically detectable tumor after 3 weeks of treatment.ConclusionsOur data demonstrates that 8-Cl-Ado treatment activates the AMPK pathway leading to autophagy induction of in breast cancer cells, eliciting, in part, its tumoricidal effects. Additionally, 8-Cl-Ado effectively inhibited in vivo tumor growth in mice. Based on this biological activity, we are planning to test 8-Cl-Ado in the clinic for patients with breast cancer.

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

Abstract 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 IC50's 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 P4-12-30: A bivalent Her2 targeting DARPin with high efficacy against Her2-low and Her2-positive tumors

Abstract The approval of Herceptin, Perjeta and Kadcyla has dramatically improved the outcome of the 15% of breast cancer patients with high expression of Her2 (Her2-positive, IHC3+). In contrast, up to 50% of women with breast cancer express low levels of Her2 (Her2-low, IHC+2 or +1) at diagnosis, and these patients do not derive benefit from approved Her2 targeted therapies. It is estimated that 5-10% of Her2-positive patients will also develop Her2-low metastases refractory to treatment. Therefore, new therapeutic strategies are needed to alleviate the high tumor burden of Her2-low patients. We generated a bivalent Designed Ankyrin Repeat Protein (DARPin) containing two anti-Her2 moieties that target unique non-overlapping epitopes on Her2 (biparatopic). The biparatopic DARPin exerts a strong pro-apoptotic response on a panel of breast and gastric cancer cell lines, which cannot be fully recapitulated by treatment with Herceptin, Perjeta or both combined. DARPin treatment of Her2-positive breast cancer BT474 cells induces apoptosis (IC50 &amp;lt; 1nM) in virtually all cells within 24 hours, whereas single or combined treatment with Herceptin and/or Perjeta reduces proliferation by 50% (IC50 = 2-4nM). The biparatopic DARPin inhibits as well the viability (via apoptosis) of Her2-low MDAMB175 cells with a 100-fold higher potency than Herceptin. Mechanistically, treatment with the anti-Her2 biparatopic DARPin inhibits both Her2 and Her3 signaling, which in turn results in the induction of apoptosis. Interestingly, cell treatment with the mix of the two individual anti-Her2 DARPin moieties does not recapitulate the apoptotic effect of the biparatopic molecule, indicating that both moieties must be linked for maximum efficacy. In vivo, the biparatopic DARPin shows efficient anti-tumor activity in established breast cancer Her2-positive xenografts and Her2-low PDX tumor models. DARPin treatment of Her2-positive BT474 xenograft in BALB/c mice results in rapid tumor regression (50% tumor shrinkage after one dose) that is sustained at least during one month after the last dose. The biparatopic DARPin strongly inhibits tumor growth as well in a Her2-low breast cancer PDX in NMRI mice, where it demonstrates superior efficacy compared to Herceptin (DARPin/Herceptin tumor volume ratio &amp;lt; 30%). In summary, biparatopic targeting of Her2 enables inhibition of both Her2 and Her3, and blocks a tumor driver and its resistance mechanism. As a result, the DARPin interferes with both the proliferation and survival of tumor cells and triggers durable anti-tumor responses in vivo. DARPin treatment of Her2 positive tumors in mice show equivalent efficacy to Herceptin with faster kinetics; an effect that is magnified in Her2-low PDX tumors where the DARPin provides superior tumor control to Herceptin. Altogether, our data demonstrate the potential for the biparatopic DARPin to surpass some of the limitations of approved Her2 targeted agents, and warrants clinical investigation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-30.

Co-delivery of thioridazine and doxorubicin using polymeric micelles for targeting both cancer cells and cancer stem cells

In this study, thioridazine (THZ), which was reported to kill cancer stem cells, was used in a combination therapy with doxorubicin (DOX) to eradicate both cancer cells and DOX-resistant cancer stem cells to mitigate the reoccurrence of the disease. Both THZ and DOX were loaded into micelles with sizes below 100 nm, narrow size distribution and high drug content. The micelles were self-assembled from a mixture of acid-functionalized poly(carbonate) and poly(ethylene glycol) diblock copolymer (PEG-PAC) and urea-functionalized poly(carbonate) (PUC) and PEG diblock copolymer (PEG-PUC). The drug-loaded mixed micelles (MM) were used to target both cancer cells and stem cells via co-delivery. Cancer stem cells were sorted by a side population assay from BT-474 and MCF-7 human breast cancer cell lines, and identified by CD44+/CD24− phenotype. The cytotoxicity of various formulations was evaluated on the sorted cancer stem cells (side population SP cells), sorted non-stem-like cancer cells (non-side population NSP cells) and unsorted cancer cells. Antitumor activity was also evaluated on BT-474 xenografts in nude mice. As compared with NSP cells, DOX suppressed SP cell growth less effectively, while THZ and THZ-MM were more effective in the inhibition of SP cells. A stronger inhibitory effect was observed on SP cells with the co-delivery of free DOX and THZ or DOX-MM and THZ-MM as compared to free DOX or DOX-MM. THZ and THZ-MM were capable of lowering the population of SP cells in unsorted cells. In the BT-474 xenografts, the co-delivery of DOX-MM and THZ-MM produced the strongest antitumor efficacy, and both THZ and THZ-MM showed strong activity against cancer stem cells. This combination therapy may provide a promising strategy for breast cancer treatment by targeting both cancer cells and cancer stem cells.

Abstract C238: Polyacetal polymer-based anti-HER2 antibody-drug conjugate employing cysteine bioconjugation through thioether linkage allows a high drug loading of dolastatin-derived payload with excellent pharmacokinetics and potent anti-tumor activity.

Abstract The application of biodegradable, polyacetal polymers to antibody-drug conjugate (ADC) design can provide numerous advantages, including significantly higher capacity for drug payload (∼20 drugs per antibody), the use of payloads with low potency that are not suitable for direct conjugation, the improvement of physicochemical properties for ADC, especially with highly hydrophobic payloads and the use of protein recognition scaffolds beyond the commonly used IgGs. The basis of this novel polyacetal polymer-based conjugation system is a hydrophilic, fully biodegradable polyacetal carrier (PHF or poly(1-hydroxymethylethylene hydroxymethylformal, or Fleximer®) modified with chemically orthogonal linkers. One linker is used to covalently attach a targeting moiety (mAb or alternative), while a second, chemically distinct linker is used to attach a drug payload and to control the mechanism and rate of drug release. Previously, we have reported potent anti-tumor activity with trastuzumab-s-Dolaflexin™, an anti-HER2 ADC composed of trastuzumab and a proprietary dolastatin derivative coupled to a Fleximer scaffold (Dolaflexin™). In that example, Dolaflexin was conjugated to the antibody through interchain cysteine residues via a hindered disulfide linkage (AACR Annual Meeting 2013 Abstract #4331). Unlike direct drug-cysteine linked ADCs that can result in destabilization of antibody by disruption of interchain disulfide bridges, we have shown that Dolaflexin conjugation via cysteines in the antibody hinge region stabilizes the resulting ADCs through the formation of inter-chain bridge structures. Trastuzumab-s-Dolaflexin ADC exhibited a prolonged plasma half-life, tumor-specific accumulation, and potent anti-tumor activity in vivo. Here, we report a novel trastuzumab-m-Dolaflexin ADC using a maleimide linker that further enhances the pharmacokinetics of the ADC and demonstrates complete regressions of established HER2+ BT-474 xenograft tumors in SCID mice even at a single dose of 2.5 mg/kg. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C238. Citation Format: Joshua Thomas, Alex Yurkovetskiy, Natalya Bodyak, Mao Yin, Patrick Conlon, Cheri Stevenson, Alex Uttard, Liu Qin, Dmitry Gumerov, Elena Ter-Ovaneysan, Michael DeVit, Laura L. Poling, Peter U. Park, Timothy B. Lowinger. Polyacetal polymer-based anti-HER2 antibody-drug conjugate employing cysteine bioconjugation through thioether linkage allows a high drug loading of dolastatin-derived payload with excellent pharmacokinetics and potent anti-tumor activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C238.

Combination of Antibody That Inhibits Ligand-Independent HER3 Dimerization and a p110α Inhibitor Potently Blocks PI3K Signaling and Growth of HER2+ Breast Cancers

We examined the effects of LJM716, an HER3 (ERBB3) neutralizing antibody that inhibits ligand-induced and ligand-independent HER3 dimerization, as a single agent and in combination with BYL719, an ATP competitive p110α-specific inhibitor, against HER2-overexpressing breast and gastric cancers. Treatment with LJM716 reduced HER2-HER3 and HER3-p85 dimers, P-HER3 and P-AKT, both in vitro and in vivo. Treatment with LJM716 alone markedly reduced growth of BT474 xenografts. The combination of LJM716/lapatinib/trastuzumab significantly improved survival of mice with BT474 xenografts compared with lapatinib/trastuzumab (P = 0.0012). LJM716 and BYL719 synergistically inhibited growth in a panel of HER2+ and PIK3CA mutant cell lines. The combination also inhibited P-AKT in HER2-overexpressing breast cancer cells and growth of HER2+ NCI-N87 gastric cancer xenografts more potently than LJM716 or BYL719 alone. Trastuzumab-resistant HER2+/PIK3CA mutant MDA453 xenografts regressed completely after 3 weeks of therapy with LJM716 and BYL719, whereas either single agent inhibited growth only partially. Finally, mice with BT474 xenografts treated with trastuzumab/LJM716, trastuzumab/BYL719, LJM716/BYL719, or trastuzumab/LJM716/BYL719 exhibited similar rates of tumor regression after 3 weeks of treatment. Thirty weeks after treatment discontinuation, 14% of mice were treated with trastuzumab/LJM716/BYL719, whereas >80% in all other treatment groups were sacrificed due to a recurrent large tumor burden (P = 0.0066). These data suggest that dual blockade of the HER2 signaling network with an HER3 antibody that inhibits HER2-HER3 dimers in combination with a p110α-specific inhibitor in the absence of a direct HER2 antagonist is an effective treatment approach against HER2-overexpressing cancers.

Abstract B046: Therapeutic targeting of integrin αvβ6 in high-risk breast cancer

Abstract Background: Integrin αvβ6 promotes migration, invasion and survival of cancer cells, however, the relevance and role of αvβ6 has yet to be elucidated in breast cancer. Methods: Protein expression of integrin subunit beta6 (β6) was measured in over 2000 breast cancers by immunohistochemistry and ITGB6 mRNA expression measured in the METABRIC dataset. Overall survival was assessed using Kaplan-Meier curves and bioinformatics statistical analyses were performed in R statistical environment v2.14.1. Using antibody (264RAD; supplied by AZ-Medimmune) blockade and siRNA knockdown of β6 in breast cell lines, the role of αvβ6 in HER2 biology (expression, proliferation, invasion, growth in vivo) was assessed by flow cytometry, MTT assays and Transwell invasion assays and xenografts, respectively. All statistical tests were two-sided. Results: High expression of either the mRNA or protein for the integrin subunit β6 correlated with very poor survival (HR=1.99, P=2.9x10-6) and increased metastases to distant sites (P=0.02). Co-expression of β6 and HER2 gave a worse prognosis (HR=3.43, P=4x10-12). HER2-driven invasion was mediated by αvβ6 in an Akt2-dependent manner. Monotherapy with 264RAD or trastuzumab, slowed growth of MCF-7/HER2-18 and BT-474 xenografts to a similar degree (P&amp;lt;0.001) but combining 264RAD with trastuzumab effectively stopped tumor growth, even in trastuzumab-resistant MCF-7/HER2-18 xenografts. Conclusions: Targeting αvβ6 with 264RAD alone or in combination with trastuzumab may provide a novel therapy for treating high-risk and trastuzumab-resistant breast cancer patients, giving hope to the 70% of women treated who have, or develop, resistance. Moreover, routine determination of the level of expression of αvβ6 on breast cancers would stratify women into higher-risk categories, requiring enhanced therapeutic intervention. Citation Format: Kate M. Moore, Gareth J. Thomas, Stephen W. Duffy, Jane Warwick, Rhian Gabe, Patrick Chou, Ian O. Ellis, Andrew R. Green, Syed Haider, Kellie Brouilette, Antonio Saha, Sabari Vallath, Rebecca Bowen, Claudia Chelala, Diana Eccles, William J. Tapper, Alastair M. Thompson, Phillip Quinlan, Lee Jordan, Cheryl Gillett, Adam Brentnall, Sheila Violette, Paul Weinreb, Jane Kendrew, Simon T. Barry, Ian R. Hart, Louise Jones, John F. Marshall. Therapeutic targeting of integrin αvβ6 in high-risk breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B046.

Neratinib overcomes trastuzumab resistance in HER2 amplified breast cancer

Trastuzumab has been shown to improve the survival outcomes of HER2 positive breast cancer patients. However, a significant proportion of HER2-positive patients are either inherently resistant or develop resistance to trastuzumab. We assessed the effects of neratinib, an irreversible panHER inhibitor, in a panel of 36 breast cancer cell lines. We further assessed its effects with or without trastuzumab in several sensitive and resistant breast cancer cells as well as a BT474 xenograft model. We confirmed that neratinib was significantly more active in HER2-amplified than HER2 non-amplified cell lines. Neratinib decreased the activation of the 4 HER receptors and inhibited downstream pathways. However, HER3 and Akt were reactivated at 24 hours, which was prevented by the combination of trastuzumab and neratinib. Neratinib also decreased pHER2 and pHER3 in acquired trastuzumab resistant cells. Neratinib in combination with trastuzumab had a greater growth inhibitory effect than either drug alone in 4 HER2 positive cell lines. Furthermore, trastuzumab in combination with neratinib was growth inhibitory in SKBR3 and BT474 cells which had acquired resistance to trastuzumab as well as in a BT474 xenograft model. Innately trastuzumab resistant cell lines showed sensitivity to neratinib, but the combination did not enhance response compared to neratinib alone. Levels of HER2 and phospho-HER2 showed a direct correlation with sensitivity to neratinib. Our data indicate that neratinib is an effective anti-HER2 therapy and counteracted both innate and acquired trastuzumab resistance in HER2 positive breast cancer. Our results suggest that combined treatment with trastuzumab and neratinib is likely to be more effective than either treatment alone for both trastuzumab-sensitive breast cancer as well as HER2-positive tumors with acquired resistance to trastuzumab.

Effect of pan-PI3K and HER2 blockade on antitumor activity in preclinical models of breast cancer resistance to trastuzumab therapy.

e13570 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). 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. Methods: 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. 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 IC50’s 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, (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) xenograft data show that the combination of GDC-0941 and T has strongly enhanced anti-tumor effect in both sensitive (82%) and resistant models (79%), something that cannot be achieved by either monotherapy. 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 activity of T therapy, as well as to prevent or delay the development of resistance.