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The development of multiple PARP inhibitors in BRCA mutant cancers has been one of the most successful implementations of molecularly targeted therapies in oncology. The recent approval of niraparib in BRCA WT Ovarian cancers demonstrates the potential for expansion of PARP inhibitors to other BRCA WT indications. The successful development of PARP inhibitors into new cancer indications will depend on using molecular biomarkers to identify tumors with a unique dependency on PARP1/2 activity. We screened a large (N=523), diverse panel of human cancer cell lines for response to three approved PARP inhibitors: olaparib, niraparib, and talazoparib. Despite differences in relative potency, there was a strong correlation between sensitive and resistant cell lines to all three compounds. In vitro experimentation revealed the inhibitors share a similar biologic mechanism of response, namely a global decrease in PARylation, as well as PARP-trapping at DNA nicks leading to G2 arrest and ultimately cell death. A suite of baseline genomic and proteomic characteristics were found to be strongly associated with response to PARP1/2 inhibition, and were largely consistent between different cancer types. Special attention was paid to BRCA1/2 variants, which represent the only approved patient selection biomarkers. Notably, neither mutations in BRCA1 nor BRCA2 were found to be associated with response to PARP1/2 inhibition in our panel, even when restricting to those with confirmed deleterious ClinVar scores. The molecular predictors of response identified in our screen may ultimately be used to develop diagnostic tools for enrollment into biomarker-enriched clinical trials to expand the use of this promising class of drug into areas of high unmet need. Triple-negative breast cancer represents one of the most intriguing spaces for development. The clinical failures in this space may be partially explained by the lack of empirical predictive biomarkers in the trial design. Our diagnostics may finally allow for the efficacious deployment of PARP inhibitors to triple-negative breast cancer. In particular we have identified a panel of biomarkers that can predict for sensitivity to PARP inhibitors in breast cancer that could be used as patient selection criteria for the expanded clinical development of these compounds into PARP-naïve patient populations with high unmet need. Citation Format: Martina SJ McDermott, Dylan F Conklin, Neil A O'Brien, Kevin Chau, Dennis J Slamon. Pan-cancer analysis of PARP inhibition reveals a suite of biomarkers that correlate with PARP1/2 activity in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-05-07.

Background: Pharmacologically targeting the HER2 oncoprotein provides clinical benefit for patients with HER2-amplified cancers. However, a significant number of patients do not respond to the currently approved HER2-targeted therapies, despite carrying the HER2-alteration. Small molecule inhibitors of HER2, that target other receptor tyrosine kinases such as EGFR (i.e. lapatinib), are approved and provide some clinical benefit but are often associated with increased toxicity. Tucatinib (ARRY-380) is an orally available, potent, highly selective small molecule inhibitor of the HER2 kinase. In this study, we assessed the in vitroand in vivoactivity of tucatinib, relative to approved HER2-targeting molecules, in a panel of molecularly characterized breast cancer cell lines. Materials and Methods:The growth inhibitory activity of tucatinib, trastuzumab and lapatinib were evaluated in a panel of 48 breast cancer cell lines molecularly characterized at baselineby genomic (array-CGH) and proteomic (Reverse Phase Protein Array; RPPA) profiling. IC50values for tucatinib and lapatinibwere determined from direct cell counts using a Cellavista Cell Imaging System. Trastuzumab activity was measured as % inhibition of cell growth at fixed concentrations. In vivoefficacy of tucatinib was assessed in cell line xenograft models of HER2+/ER- and HER2+/ER+ breast cancers as a single agent or in combination with targeted therapies for breast cancer. Results: A broad range of IC50values (3.2nM to >10μM), was seen for tucatinib with a high degree of selectivity for the HER2-amplfied sub-type. High levels of total and phosphorylated HER2 (pHER2) accompanied by high levels of pEGFR and pHER3 enriched for sensitivity to tucatinib, confirming that HER2-driven cancers may be uniquely sensitive to tucatinib. The response profile for lapatinib was less clean, with responses also observed in HER2-low/EGFR-high cell lines. Sensitivity to tucatinib was also observed in HER2-amplified cell lines that were either de novoor acquired resistant to trastuzumab. Single agent tucatinib induced tumor regressions in a xenograft model of HER2+/ER+ breast cancer. Tumor regressions were further enhanced by combination with trastuzumab. The combination of tucatinib plus trastuzumab was as efficacious and better tolerated than trastuzumab plus docetaxel or trastuzumab plus pertuzumab plus docetaxel. The triple combination of tucatinib plus hormonal blockade (fulvestrant) and CDK4/6 inhibition (abemaciclib) also induced robust tumor regressions, without significant body weight loss. Discussion: These preclinical data highlight the potential of the HER2-selective small molecule inhibitor, tucatinib, to provide benefit to patients with HER2-amplifed cancers. Furthermore, our biomarker analysis of response to tucatinib has identified a HER2-driven signature within the HER2-amplfied sub-type that selects for sensitivity to tucatinib. Selecting patients based on this profile may further enrich for individuals most likely to benefit from tucatinib-based therapies. Citation Format: O'Brien NA, Conklin D, McDermott M, Luo T, Ayala R, Issakhanian S, Salgar S, Hurvitz S, Slamon DJ. The small molecule inhibitor of HER2, tucatinib, has potent and highly selective activity in preclinical modes of HER2-driven cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-17-11.

BackgroundCombined targeting of CDK4/6 and ER is now the standard of care for patients with advanced ER+/HER2− breast cancer. However, acquired resistance to these therapies frequently leads to disease progression. As such, it is critical to identify the mechanisms by which resistance to CDK4/6-based therapies is acquired and also identify therapeutic strategies to overcome resistance.MethodsIn this study, we developed and characterized multiple in vitro and in vivo models of acquired resistance to CDK4/6-based therapies. Resistant models were screened by reverse phase protein array (RPPA) for cell signaling changes that are activated in resistance.ResultsWe show that either a direct loss of Rb or loss of dependence on Rb signaling confers cross-resistance to inhibitors of CDK4/6, while PI3K/mTOR signaling remains activated. Treatment with the p110α-selective PI3K inhibitor, alpelisib (BYL719), completely blocked the progression of acquired CDK4/6 inhibitor-resistant xenografts in the absence of continued CDK4/6 inhibitor treatment in models of bothPIK3CAmutant and wild-type ER+/HER2− breast cancer. Triple combination therapy against PI3K:CDK4/6:ER prevented and/or delayed the onset of resistance in treatment-naive ER+/HER2− breast cancer models.ConclusionsThese data support the clinical investigation of p110α-selective inhibitors of PI3K, such as alpelisib, in patients with ER+/HER2− breast cancer who have progressed on CDK4/6:ER-based therapies. Our data also support the investigation of PI3K:CDK4/6:ER triple combination therapy to prevent the onset of resistance to the combination of endocrine therapy plus CDK4/6 inhibition.

The cyclinD:CDK4/6:Rb axis is dysregulated in a variety of human cancers. Targeting this pathway has proven to be a successful therapeutic approach in ER+ breast cancer. In this study, in vitro and in vivo preclinical breast cancer models were used to investigate the expanded use of the CDK4/6 inhibitor, abemaciclib. Using a panel of 44 breast cancer cell lines, differential sensitivity to abemaciclib was observed and was seen predominately in the luminal ER+/HER2− and ER+/HER2+ subtypes. However, a subset of triple-negative breast cancer (TNBC) cell lines with intact Rb signaling were also found to be responsive. Equivalent levels of tumor growth inhibition were observed in ER+/HER2−, ER+/HER2+ as well as biomarker selected TNBC xenografts in response to abemaciclib. In addition, abemaciclib combined with hormonal blockade and/or HER2-targeted therapy induced significantly improved antitumor activity. CDK4/6 inhibition with abemaciclib combined with antimitotic agents, both in vitro and in vivo, did not antagonize the effect of either agent. Finally, we identified a set of Rb/E2F-regulated genes that consistently track with growth inhibitory response and constitute potential pharmacodynamic biomarkers of response to abemaciclib. Taken together, these data represent a comprehensive analysis of the preclinical activity of abemaciclib, used alone or in combination, in human breast cancer models. The subtypes most likely to respond to abemaciclib-based therapies can be identified by measurement of a specific set of biomarkers associated with increased dependency on cyclinD:CDK4/6:Rb signaling. These data support the clinical development of abemaciclib as monotherapy or as a combination partner in selected ER+/HER2−, HER2+/ER+, and TNBCs. Mol Cancer Ther; 17(5); 897–907. ©2018 AACR.

Everolimus (RAD001, Afinitor(®)) is an oral, selective mTOR inhibitor recently approved by the US-FDA in combination with exemestane for treatment of hormone receptor positive advanced breast cancer. To date, no molecular predictors of response to everolimus in breast cancer have been identified. We hypothesized predictive markers could be identified using preclinical models. Using a molecularly characterized panel of human breast cancer and immortalized breast epithelial cell lines, we determined sensitivity to everolimus alone or in combination with ER- or HER2- targeted therapy. Gene expression microarrays and comparative genomic hybridization were performed on the cell lines to identify predictors of response to everolimus. Among 13 everolimus-sensitive cell lines, 10/13(77 %) were luminal, while in 26 resistant cell lines, 16/26(62 %) were non-luminal, and 10/26(38 %) were luminal. Only 3/24 non-luminal lines were sensitive, two of which were HER2+. Everolimus enhanced the anti-proliferative effect of both tamoxifen (TAM) and fulvestrant (FUL) in ER+ breast cancer cell lines, as well as trastuzumab in HER2+ cell lines. Everolimus + FUL but not everolimus + TAM reversed acquired resistance to TAM. Everolimus inhibited mTOR in tested cell lines by decreasing S6 phosphorylation, mediating its anti-proliferative effect by G0/G1 cell cycle arrest and induction of apoptosis. Chromosomal amplifications of AURKA (p value = 0.04) and HER2 (p value = 0.03) were each associated with increased sensitivity to everolimus. Transcript expression microarrays identified GSK3A, PIK3R3, KLF8, and MAPK10 among the genes overexpressed in sensitive luminal lines, while PGP, RPL38, GPT, and GFAP were among the genes overexpressed in resistant luminal cell lines. These preclinical in vitro data provide further support for continued clinical development of everolimus in luminal (ER+ or HER2+) breast cancer in combination with targeted therapies. We identified several potential molecular markers associated with response to everolimus that will require validation in clinical material.

Addition of CDK4/6 inhibitors such as palbociclib, ribociclib or abemaciclib to endocrine-based therapies significantly improves progression-free and in some subgroups, overall survival in patients with advanced estrogen receptor-positive (ER+) breast cancer. However, acquired resistance to CDK4/6 inhibitors remains a significant unmet clinical need. It is critical to ascertain the mechanisms by which tumor cells evade CDK4/6 based therapy. In this study we screen multiple in vitro and in vivo models of acquired resistance to CDK4/6 inhibitors to identify potential resistance/escape pathways and targets for pharmaceutical intervention to overcome resistance. ER+ breast cancer cell lines of diverse molecular backgrounds were conditioned to acquire resistance to CDK4/6 inhibitors through either long-term culture in the presence of clinically relevant concentrations of inhibitors or as cell line xenografts treated through progression on a CDK4/6 inhibitor plus fulvestrant. Baseline and pharmacodynamic changes in cell signaling were measured using reverse phase protein array (RPPA) and RNAseq analysis. Acquired resistance to CDK4/6 inhibitors was associated with decreases in phosphorylated-Rb (pRb) and ER-alpha protein and increases in pAKT and pS6 relative to isogenic controls. The p110α-selective PI3K-inhibitor, alpelisib, in combination with fulvestrant or ribociclib/fulvestrant blocked pAKT signaling in xenografts progressing on palbociclib/fulvestrant and induced significant tumor regressions. Apelisib plus fulvestrant also produced robust anti-tumor responses in xenografts progressing on ribociclib/fulvestrant. Triple combination treatment with ribociclib/alpelisib/fulvestrant induced significant regressions in resistant tumors and in treatment naïve models, where complete tumor regressions occurred regardless of PIK3CA mutation status. Regressions were maintained for >9 weeks post withdrawal of treatment, indicating that therapeutic resistance may be prevented by this triple combination approach. RPPA analysis of responding tumors identified sustained inhibition of both PI3K- and CDK4/6:Rb-pathway signaling accompanied by activation of pro-apoptotic proteins. These data support clinical investigation of targeting PI3K with alpelisib in breast cancers progressing on CDK4/6 based therapies and investigation of upfront triple combination therapy prior to acquisition of resistance to CDK4/6. Citation Format: Neil A. O'Brien, Martina SJ McDermott, Dylan F. Conklin, Alex Gaither, Tong Luo, Raul Ayala, Suruchi Salgar, Emmanuelle DiTomaso, Naveen Babbar, Faye Su, Sara A. Hurvitz, Ronald Linnartz, Kristine Rose, Samit Hirawat, Dennis J. Slamon. Targeting activated PI3K/mTOR signaling overcomes resistance to CDK4/6-based therapies in preclinical ER+ breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3825.

HSP90 enables the activation of many client proteins of which the most clinically validated is HER2. NVP-AUY922, a potent HSP90 inhibitor, is currently in phase II clinical trials. To explore its potential clinical use in HER2-amplified breast and gastric cancers, we evaluated the effect of AUY922 alone and in combination with trastuzumab in both trastuzumab-sensitive and -resistant models. A panel of 16 human gastric and 45 breast cancer cell lines, including 16 HER2-amplified (3 and 13, respectively) cells, was treated with AUY922 over various concentrations. In both breast and gastric cancer, we used cell lines and xenograft models with conditioned trastuzumab-resistance to investigate the efficacy of AUY922 alongside trastuzumab. Effects of this combination on downstream markers were analyzed via Western blot analysis. AUY922 exhibited potent antiproliferative activity in the low nanomolar range (

The human EGF (HER) family of receptors has been pursued as therapeutic targets in breast cancer and other malignancies. Trastuzumab and lapatinib are standard treatments for HER2-amplified breast cancer, but a significant number of patients do not respond or develop resistance to these drugs. Here we evaluate the in vitro activity of dacomitinib (PF-00299804), an irreversible small molecule pan-HER inhibitor, in a large panel of human breast cancer cell lines with variable expression of the HER family receptors and ligands, and with variable sensitivity to trastuzumab and lapatinib. Forty-seven human breast cancer and immortalized breast epithelial lines representing the known molecular subgroups of breast cancer were treated with dacomitinib to determine IC50 values. HER2-amplified lines were far more likely to respond to dacomitinib than nonamplified lines (RR, 3.39; P < 0.0001). Furthermore, HER2 mRNA and protein expression were quantitatively associated with response. Dacomitinib reduced the phosphorylation of HER2, EGFR, HER4, AKT, and ERK in the majority of sensitive lines. Dacomitinib exerted its antiproliferative effect through a combined G0–G1 arrest and an induction of apoptosis. Dacomitinib inhibited growth in several HER2-amplified lines with de novo and acquired resistance to trastuzumab. Dacomitinib maintained a high activity in lines with acquired resistance to lapatinib. This study identifies HER2-amplified breast cancer lines as most sensitive to the antiproliferative effect of dacomitinib and provides a strong rationale for its clinical testing in HER2-amplified breast cancers resistant to trastuzumab and lapatinib. Mol Cancer Ther; 11(9); 1978–87. ©2012 AACR.

The selective cyclin dependent kinases 4 and 6 (CDK-4/6) inhibitor, palbociclib, has recently been approved in combination with endocrine-based therapies for the treatment of advanced ER+/HER2- breast cancer (BC). Despite the improvements in progression-free and overall survivals, a significant number of patients on CDK-4/6 therapy will go on to develop progressive disease. As such, it is critical to identify the mechanisms by which tumor cells evade CDK-4/6 targeted therapy and to develop therapeutic strategies to overcome resistance. In this study, we evaluated the efficacy of a p110α-selective PI3K inhibitor, alpelisib (BYL719) and the mTORC1 specific inhibitor, everolimus (RAD001), in ER+/HER2- xenograft tumors conditioned in vitro and in vivo to acquire resistance to combined palbociclib + endocrine-based therapy. The EFM19 (ER+/HER2-, PIK3CA mt), MDA134 (ER+/HER2-, PIK3CA wt), and MDA361 (ER+/HER2+, PIK3CA mt) BC cell lines were conditioned to acquire palbociclib resistance through long-term culture in the presence of increasing concentrations of drug. Baseline total/phosphoprotein levels of over 280 cancer-associated analytes were measured in the parental and resistant cell lines by Reverse Phase Protein Array (RPPA). In vivo resistant models were developed from xenografts of the MCF7 and HCC1500 ER+ BC cell lines, treated until progression with palbociclib (75 mg/kg) + fulvestrant (5 mg/mse). EFM19-PR cells maintained palbociclib resistance in vivo and were included in the xenograft studies. Resistance to palbociclib was confirmed in vitro by a shift in growth inhibitory IC50 from an average of less 50nM to over 1 µM. Significantly reduced Rb and ER protein levels were detected in each of these palbociclib resistant cell lines by RPPA. Cross-resistance to an alternative CDK-4/6 inhibitor (ribociclib) was found in in vitro studies. In addition, switching treatment of the xenografts progressing on palbociclib + fulvestrant to ribociclib (75mg/kg) + fulvestrant did not impact tumor progression. However, when progressing tumors were switched to BYL719 (30mg/kg) + fulvestrant or RAD001 (10mg/kg) + fulvestrant, sustained tumor regression was observed for over 45 days of treatment in every model tested. The triplet combination of ribociclib + fulvestrant + BYL719 or RAD001 provided marginal additional benefit over the doublet combination of BYL719 or RAD001 + fulvestrant. RPPA analysis of xenograft tissue collected from these studies will help to identify additional molecular alterations involved in resistance to CDK-4/6 targeted therapy. These preclinical data indicate that acquired resistance to CDK-4/6 inhibition occurs through a loss of dependence on Rb-signaling. However, targeting an alternative pathway like PI3K/mTOR with molecules such as BYL719 or RAD001 may be a viable strategy for further clinical investigation. Citation Format: Neil A. O'Brien, Dylan Conklin, Tong Luo, Raul Ayala, Shawnt Issakhanian, Ondrej Kalous, Erika Von Euw, Sara A. Hurvitz, Emmanuelle DiTomaso, Faye Su, Ronald Linnartz, Stefan Scherer, Samit Hirawat, Dennis J. Slamon. Anti-tumor activity of the PI3K/mTOR pathway inhibitors alpelisib (BYL719) and everolimus (RAD001) in xenograft models of acquired resistance to CDK-4/6 targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4150. doi:10.1158/1538-7445.AM2017-4150

The cyclin D:CDK-4/6:Rb axis is dysregulated in a number of different cancers and is implicated in resistance to hormonal therapy in breast cancer. Pharmacologically targeting cyclin dependent kinase 4 and 6 (CDK4 and 6) has proven to be a successful therapeutic approach in ER+ breast cancer (BC). This study aimed to identify the molecular subtypes of BC that are sensitive to the novel CDK4 and 6 inhibitor, abemaciclib, and identify the best combination strategies for the clinical development. Growth inhibition activity of abemaciclib was assessed in a panel of 46 BC cell lines molecularly characterized by genomic, transcriptomic and proteomic profiling. IC50 values were determined from direct cell counts using a Z1-particle counter. In vivo activity of abemaciclib was assessed in cell line xenograft models of ER+ and HER2+/ER+ BCs. For ER+ BC, mice were treated daily with clinically achievable doses of abemaciclib (50-75 mg/kg) as a single agent or in combination with tamoxifen or fulvestrant. Combinations with trastuzumab, docetaxel and tamoxifen were assessed in the HER2+/ER+ xenografts. Sensitivity to abemaciclib was observed predominately in multiple luminal BC cell lines and a small subset of triple negative cell lines that had intact Rb signaling. Activating mutations in PIK3CA also marked for abemaciclib sensitivity. Abemaciclib potentiated the anti-proliferative effects of cytotoxic/anti-mitotic agents when given simultaneously or 48 hours prior to treatment in vitro. Significant tumor growth inhibition (TGI) was observed with single agent abemaciclib in the ER+ BC cell line xenografts. In ZR751 xenografts, the addition of either tamoxifen or fulvestrant to abemaciclib induced complete inhibition of tumor growth for the 12 weeks of treatment. In the MCF7 model, treatment was withdrawn after five weeks, which triggered tumor regrowth in each of the single agent arms. However, complete responses were maintained in the combination arms for a further six weeks post drug withdrawal. In HER2 amplified xenografts, abemaciclib single agent treatment induced significant TGI in trastuzumab sensitive and resistant xenografts, and combination with trastuzumab further increased this anti-tumor effect. The addition of tamoxifen to this combination induced a further increment in TGI. Consistent with the in vitro findings, the combination of abemaciclib and the anti-mitotic agent docetaxel was not antagonistic in vivo, and the addition of docetaxel to the triple combination of abemaciclib, trastuzumab, and tamoxifen induced the most efficacy of any of the treatment arms tested. Combinations were well tolerated in animals. These data highlight the potential of abemaciclib to have single agent activity in addition to combined activity with anti-mitotic or targeted therapies for breast cancer. Citation Format: Neil A. O’Brien, Dylan Conklin, Tong Luo, Ondrej Kalous, Erika von Euw, Sara A. Hurvitz, Richard P. Beckmann, Colleen Mockbee, Dennis J. Slamon. Preclinical activity of abemaciclib as a single agent or in combination with anti-mitotic or targeted therapies for 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 2828.

Introduction: Notch-3 overexpression has been implicated in the development of breast cancer (BC) and is associated with poor outcomes. A critical challenge to eliminating treatment resistance in breast cancer likely relates to the presence of cancer stem cells (CSCs) that maintain the ability to differentiate and divide indefinitely. We postulate that targeted eradication of CSCs is possible using a Notch3 antibody drug conjugate (ADC) without irreversibly reducing stem cell viability in vital normal tissues. PF-06650808, is an ADC comprised of a humanized anti-Notch-3 antibody linked to an auristatin-based cytotoxic agent. To better understand the therapeutic index of targeting Notch-3, we evaluated PF-06650808 across a large panel of BC lines and normal cells and correlated response with Notch-3 levels. PF-06650808 was also evaluated in a murine BC xenograft model. Methods: Response to PF-06650808 and control ADC was evaluated across a panel of BC and normal cell lines by a 2D proliferation assay. Notch-3 mRNA expression was measured by flow cytometry (FC) and RPPA. MDA-MB-468 (triple negative BC, TNBC) tumor bearing mice were randomized into 4 arms of 8 mice and treated with 3 mg/kg PF-06650808 or control-ADC (days 0, 4, 8 & 12), 10 mg/kg docetaxel (q week) or vehicle control. Results: High expression of Notch-3 was detected in multiple BC cell lines by RPPA and FC. BC cell lines with elevated levels of Notch-3 were sensitive to PF-06650808 (HCC1187, MDA-MB-468, HCC1143, HCC70, EFM-19, HCC202). Responders were also enriched for TNBC. All normal cell lines were resistant to PF-06650808 ADC. When treated with a control ADC against a non-relevant target, all cell lines exhibited IC50s between 5-50ug/ml, indicating that the sub-0.5ug/ml responses seen with the Notch-3 ADC were target-dependent. Durable complete tumor regressions were observed in PF-06650808-treated mice bearing MDA-MB-468 TNBC cell line xenografts. Conclusions: Sensitivity to a novel anti-Notch-3-ADC is associated with high expression of Notch-3 in BC cell lines. Normal cells are resistant to PF-06650808, possibly predicting a better therapeutic index than seen with other Notch inhibitors. Xenograft studies evaluating the in vivo efficacy of PF-06650808 in a panel of xenografts with varying levels of Notch-3 expression will be presented. Ongoing experiments are exploring the potency of PF-06650808 on CSCs. Our data will help identify breast cancer subtypes most likely to respond to a Notch3-ADC based on high tumor/normal target concentration as well as its effects on CSCs. Citation Format: Sara A. Hurvitz, Erika von Euw, Neil O’Brien, Dylan Conklin, Chuhong Hu, Jiaying Zhuo, Alice Zhao, Frank Calzone, Hsiao-Wang Chen, Judy Dering, Ken Geles, Puja Sapra, Dennis J. Slamon. Preclinical evaluation of targeting Notch-3 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 1206.

BACKGROUND: Aurora kinases (AK) A and B play essential roles in multiple stages of mitosis and are frequently overexpressed in a subset of human cancers, including ovarian cancer (OC). AMG 900, a potent and highly selective small molecule inhibitor of AKs, showed promising single-agent activity in heavily pretreated patients with advanced OC in a Phase 1b clinical trial. In this study, we report the preclinical effects of AMG 900 in a panel of well-characterized human cancer cell lines representing clinically-relevant OC subtypes. METHODS: The anti-proliferative effects of AMG 900 were evaluated using a 5-day cell count assay. Cell lines were classified as sensitive to AMG 900 when lethality was > 15% at 10 nM. Molecular markers were profiled including TP53 mutation status, AURKA, CCNE1, MYC copy number, and p53, p21 and cyclin E1 protein by reverse phase protein array. Flow cytometry and imaging methods were used to evaluate the mechanism of action of AMG 900 alone and in combination with chemotherapy. The combination of AMG 900 plus docetaxel was evaluated in an IGROV-1 ovarian endometrioid carcinoma xenograft model. RESULTS: One third of the cell lines (11 of 35) were classified as sensitive to AMG 900 and showed enrichment for TP53 mutations and serous OC subtype. However, 10 of 24 resistant cell lines harbored TP53 mutations, indicating that TP53 mutational status alone was not sufficient for predicting AMG 900 sensitivity. Inhibition of AK activity by AMG 900 in OC cells resulted in aborted cell division leading to polyploidy and cell death (suggestive of aurora-B dominant phenotype). Re-plating of remnant cells after AMG 900 treatment showed an attenuation of cell regrowth, where TP53mut IGROV-1 cells showed minimal regrowth compared to TP53wt OVCAR-5 cells. AMG 900 inhibited proliferation at low nanomolar concentrations in the majority of OC cell lines and enhanced the effects of paclitaxel, carboplatin, and doxorubicin in IGROV-1 cells. In tumor-bearing mice, administration of AMG 900 at 7.5 mg/kg (PO) for two days per week or docetaxel at 10 mg/kg (IP) weekly for four cycles significantly inhibited the growth of IGROV-1 tumor xenografts (P < .0001 vs. vehicle alone). Notably, co-administration of AMG 900 with docetaxel enhanced efficacy and induced a delay in tumor regrowth compared to docetaxel alone. Single-agent-treated mice showed minimal body weight loss (BWL), whereas combination-treated mice showed moderate BWL (average < 10%) that was largely reversible (2 of 12 animals removed due to toxicity). CONCLUSIONS: AMG 900 alone or in combination with chemotherapy such as paclitaxel may be a promising clinical strategy to treat patients with ovarian cancer. Citation Format: Ondrej Kalous, Dylan Conklin, Kanthinh Manivong, William Wayne, Kelly Hanestad, Jude Canon, Robert Loberg, Gregory Friberg, Erick Gamelin, Florian D. Vogl, Gloria Juan, Angela Coxon, Dennis Slamon, Richard Finn, Marc Payton. Preclinical characterization of AMG 900, a pan-aurora kinase inhibitor, alone and in combination with taxanes in ovarian 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 3008.

1099 Background: An urgent problem in breast cancer treatment is the identification and validation of a targeted therapy for triple-negative breast cancer (TNBC), ie, breast cancer negative for ER, PR and HER2. Neratinib is a low molecular weight irreversible pan-HER inhibitor that selectively targets the tyrosine kinase activity of EGFR, HER2 and HER4. To-date, neratinib has been exclusively investigated in HER2-positive tumors. However, as neratinib also targets EGFR expression, we hypothesised that it may also have clinical efficacy in TNBC. Methods: The anti-proliferative effects of neratinib were investigated in 14 TNBC cell lines. To identify potential biomarkers of response, we examined the expression of baseline EGFR, HER2, HER3 and HER4 using reverse phase protein array (RPPA) analysis and flow cytometry analysis. Results: IC50 values for neratinib across the 14 TNBC cell lines ranged from 0.06 µM to 1.9 µM. However, basal A-type cells were significantly more sensitive to neratinib than basal B-t...

Background: The addition of the selective mTORC1 inhibitor everolimus (RAD001/Afinitor®), to exemestane significantly prolongs progression free survival in patients with advanced hormone receptor (ER+) positive breast cancer. However, despite these improved outcomes, the majority of patients that achieve an initial clinical benefit will eventually go on to develop progressive disease. In order to prevent or delay the onset of this acquired resistance, it is essential to identify the mechanisms by which continued proliferation is being driven in these resistant tumors. In this study, we used reverse phase protein array (RPPA) technology to compare cell signaling in ER+ breast cancer cell line xenografts that were responsive to everolimus versus those that had acquired resistance to everolimus. Materials and Methods: Xenograft models were established from three ER+ breast cancer cell lines containing different molecular alterations that confer activated PI3K/mTOR signaling; MCF7 and KPL-1 (both PIK3CA mutant) and ZR75-1 (PTEN-null). Mice were treated daily with either vehicle or 10 mg/kg everolimus until progression. Snap frozen tissue samples (n = 3) were collected from vehicle control tumors, from everolimus resistant tumors and from everolimus responsive tumors. Whole cell lysates were prepared from tumor tissues and processed for RPPA analysis. A customized panel of 27 known cancer/PI3K pathway associated signaling proteins were selected for the RPPA analysis. Results: In the vehicle controls, significantly higher levels of pAKT, pPRAS40 and p4EBP1 were detected in the PTEN-null ZR75-1 tumors compared to the PIK3CA mutant tumors, indicating potential variations in intensity of PI3K/AKT pathway signaling depending on the initiating molecular alteration. Complete inhibition of tumor proliferation was observed in response to everolimus in all 3 xenograft models for up to 7-weeks of treatment, until tumors showed signs of progression. Significantly lower S6 phosphorylation was observed in tumors responding to everolimus, which was accompanied by feedback activation of pAKT. Significant inhibition of S6 phosphorylation was also observed in each of the ZR75-1 and MCF7 tumors that were progressing on everolimus, indicating that although the tumor cells have acquired resistance to the anti-proliferative activity of everolimus, mTORC1 signaling is still being successfully blocked. Feedback activation of pAKT was observed in the ZR75-1 resistant model, however no feedback activation of AKT occurred in the MCF7 resistant model. The KPL-1 model showed the expected loss of S6 pathway inhibition upon resistance. No significant signaling changes were observed in any of the other signaling proteins measured, including ER, HER2, EGFR, MEK and ERK. Ongoing studies to measure the mRNA expression changes in these tumors using mRNA microarrays may identify signaling pathways that are driving the proliferation of these resistant tumors. Discussion: These preclinical data show that multiple pathways of resistance can develop in response to long-term everolimus treatment and resistance can occur despite continued inhibition of PI3K/mTOR signaling. Data from mRNA microarray experiments will be presented at the time of the meeting. Citation Format: Neil A O'Brien, Dylan Conklin, Michaela Ching, Luo Tong, Raul Ayala, Shawnt Issakhanian, Sara A Hurvitz, Emmanuelle di Tomaso, Ronald Linnartz, Richard S Finn, Samit Hirawat, Dennis J Slamon. Acquired resistance to everolimus occurs independently of mTORC1 inhibition in preclinical in vivo models of ER+ breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-04.

1054 Background: Currently, one of the most urgent problems in breast cancer therapeutics is the validation of targeted therapy for triple-negative breast cancer (TNBC). Neratinib is an irreversible small molecule inhibitor that targets the kinase activity of EGFR, HER2 and HER4. As one of these targets, EGFR, is expressed at high levels in a subset of TNBC, we hypothesised that neratinib is a potential treatment for at least some patients with TNBC. Methods: The antiproliferative effects of neratinib were investigated in a panel of 35 breast cancer cells lines including 9 luminal, 12 HER2-positive and 14 triple-negative (TN). Baseline levels of total HER proteins were determined using flow cytometry. Results: IC50 values for neratinib across the panel of breast cancer cell lines ranged from < 0.001 - 1.9 µM. HER2-positive cell lines were significantly more sensitive to neratinib than HER2-negative cells (p = 0.008, Student’s t-test). For the 14 TN cell lines, IC50 values ranged from 0.03 – 1.9 µM. TN cell lines defined as basal-type A were significantly more sensitive to neratinib than those designated basal-type B (p = 0.02, Student’s t-test). Treatment with neratinib resulted in a reduction in HER-family signalling as detected by decreased Akt and ERK phosphorylation. Consistent with the finding that basal type A TN cells were more sensitive to neratinib than type B, significantly higher levels of EGFR expression were found in the A subtype compared to the B subtype (p = 0.02, Student’s t-test). To determine a possible predictive marker of response for neratinib, levels of all 4 HER proteins were related to response in the basal A subgroup. Baseline levels of HER3 expression were found to correlate significantly with response to neratinib (p = 0.02, r = -0.875, n = 7, Spearman), i.e., cell lines with the highest levels of HER3 expression had the lowest IC50values within this cohort. Conclusions: Our results suggest that neratinib is a potential novel therapeutic option for patients with basal A type triple negative breast cancer. HER3 may act as a predictive marker of response for this subgroup of patients. Acknowledgements: The authors thank SFI for funding MTCI (SRC award, 08/SRC/B1410 to MTCI) and an SFI Short Term Travel Fellowship to MM, for funding this work.

Background: Heat Shock Protein 90 (HSP90) enables the activation of a large number of client proteins of which the most clinically validated is HER2. NVP-AUY922 is a a highly potent, non-geldamycin HSP90 inhibitor that has shown preliminary activity in HER2 positive metastatic breast cancer. To explore its potential clinical utility specifically in HER2 amplified gastric and breast cancers, we evaluated the effect of AUY922 alone and in combination with trastuzumab in both trastuzumab-sensitive and resistant in vitro and in vivo models. Methods: A panel of 16 human gastric and 45 breast cancer cell lines, with 3 and 13 HER2-amplified lines respectively, were exposed in vitro to AUY922 over various concentrations to generate dose response curves. In both breast and gastric cancer, we used cell lines and xenograft models with conditioned trastuzumab-resistance to investigate the efficacy of AUY922 alongside trastuzumab. In all in vivo experiments, trastuzumab was dosed at 10 mg/kg (IP; twice/week) and AUY922 at 50 mg/kg, (IP; 5 days on, 2 days off). Effects of this combination on downstream markers (HER2, AKT, ERK) were analyzed via western blot. Results: AUY922 was found to have potent anti-proliferative activity in the low nanomolar range (< 40 nM) for 59 of the 61 gastric and breast cancer cell lines tested. Additionally, in both gastric and breast cancer, HER2-amplified cells expressed greater sensitivity to the compound when compared to the HER2-negative cells. In conditioned trastuzumab-resistant cell line models, AUY922 continued to have potent activity and exhibited a synergistic effect with trastuzumab. The in vitro combination also induced greater decreases in HER2, induced a G0/G1 cell cycle arrest, and increased the rates of apoptosis. In a conditioned trastuzumab-resistant gastric cancer in vivo model (N87-Res), the combination of AUY922 and trastuzumab showed greater anti-tumor efficacy than either drug alone. Conclusions: These data suggest that AUY922 in combination with trastuzumab has unique efficacy in trastuzumab-resistant models. The combination of HSP90 inhibitors and trastuzumab represents a novel approach to the treatment of HER2 amplified cancers. Clinical trials combining AUY922 and trastuzumab in HER2 amplified breast and gastric cancers are currently ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3668. doi:1538-7445.AM2012-3668