Neil O’Brien

Current approaches and future directions in the treatment of HER2-positive breast cancer

Human epidermal growth factor receptor 2 (HER2), a member of the ErbB family of transmembrane receptor tyrosine kinases, is amplified in 20-30% of invasive breast cancers. HER2 amplification is associated with metastasis and reduced survival. Two HER2-directed therapies have been approved by the United States Food and Drug Administration for the treatment of HER2-overexpressing breast cancer: trastuzumab, a humanized monoclonal antibody against the extracellular portion of HER2; and lapatinib, a dual HER2- and epidermal growth factor receptor-specific tyrosine kinase inhibitor. Despite the improvement in overall survival with the addition of HER2-targeted agents to chemotherapy, many patients do not benefit from these agents because of inherent resistance. In addition, many patients who achieve an initial response eventually acquire drug resistance. Currently, several mechanisms of resistance have been described, including mutations in other signaling pathways, expression of a truncated form of HER2, receptor crosstalk, and autophagy. There are several approaches under study to target these pathways of resistance, including blocking PI3 kinase and mammalian target of rapamycin signaling, blocking neoangiogenesis and the vascular endothelial growth factor axis, using monoclonal antibody targeting of the HER2 dimerization site, and using HER2 monoclonal antibody-drug conjugates. Here we will review the current scientific rationale for these agents and how combinations of these agents may yield additive or synergistic effects and lead to improved outcomes for patients with HER2-amplified breast cancer.

PP2A inhibition overcomes acquired resistance to HER2 targeted therapy

BackgroundHER2 targeted therapies including trastuzumab and more recently lapatinib have significantly improved the prognosis for HER2 positive breast cancer patients. However, resistance to these agents is a significant clinical problem. Although several mechanisms have been proposed for resistance to trastuzumab, the mechanisms of lapatinib resistance remain largely unknown. In this study we generated new models of acquired resistance to HER2 targeted therapy and investigated mechanisms of resistance using phospho-proteomic profiling.ResultsLong-term continuous exposure of SKBR3 cells to low dose lapatinib established a cell line, SKBR3-L, which is resistant to both lapatinib and trastuzumab. Phospho-proteomic profiling and immunoblotting revealed significant alterations in phospho-proteins involved in key signaling pathways and molecular events. In particular, phosphorylation of eukaryotic elongation factor 2 (eEF2), which inactivates eEF2, was significantly decreased in SKBR3-L cells compared to the parental SKBR3 cells. SKBR3-L cells exhibited significantly increased activity of protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates eEF2. SKBR3-L cells showed increased sensitivity to PP2A inhibition, with okadaic acid, compared to SKBR3 cells. PP2A inhibition significantly enhanced response to lapatinib in both the SKBR3 and SKBR3-L cells. Furthermore, treatment of SKBR3 parental cells with the PP2A activator, FTY720, decreased sensitivity to lapatinib. The alteration in eEF2 phosphorylation, PP2A activity and sensitivity to okadaic acid were also observed in a second HER2 positive cell line model of acquired lapatinib resistance, HCC1954-L.ConclusionsOur data suggests that decreased eEF2 phosphorylation, mediated by increased PP2A activity, contributes to resistance to HER2 inhibition and may provide novel targets for therapeutic intervention in HER2 positive breast cancer which is resistant to HER2 targeted therapies.

Use of a Panel of Novel Genes for Differentiating Breast Cancer from Non-Breast Tissues

Existing serum markers for breast cancer such as CA 15-3, BR 27.29 and CEA lack sensitivity and specificity. The aim of this study was to evaluate the value of new putative breast-specific markers for differentiating breast cancer from non-breast tissues. Expression of mammaglobin A (MGA), B726P, small breast epithelial mucin (SBEM) and MUC1 was measured by RT-PCR. MGA mRNA was detected in 86/162 (60%) breast cancers but in only 1/32 (3%) non-breast tissues; B726P was detected in 44/108 (41%) breast cancers but in none of 20 non-breast tissues, while SBEM was present in 52/103 (51%) breast cancers but in only 1/26 non-breast cancer tissues. In contrast to these novel markers, the established breast cancer marker MUC1 was detected in 72/99 (73%) breast cancers and in 22/32 (59%) of non-breast tissues. Combining MGA with B726P separated breast cancer from non-breast tissue with a sensitivity of 71% and a specificity of 95% while combining MGA with SBEM differentiated breast cancer from non-breast tissues with a sensitivity of 76% and a specificity of 89%. Genes such as MGA, B726P and SBEM that are expressed relatively exclusively in breast tissue are potential new markers for breast cancer.

Development of acquired resistance to lapatinib may sensitise HER2-positive breast cancer cells to apoptosis induction by obatoclax and TRAIL

BackgroundLapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells.MethodsWe examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer.ResultsIn this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells.ConclusionsOur findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.

Abstract 2828: Preclinical activity of abemaciclib as a single agent or in combination with anti-mitotic or targeted therapies for breast cancer

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.

Abstract 1206: Preclinical evaluation of targeting Notch-3 in breast cancer

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.

Abstract 2736: Regulation of cytokinesis by polo-like kinase 4

Introduction. The serine/threonine mitotic kinase, polo-like kinase 4 (PLK4), is known to play a critical role in centrosome duplication in preparation for cell division. Based on our preliminary observations with a PLK4 kinase inhibitor, we investigated the possibility that PLK4 may also play a role in regulation of cytokinesis. Experimental Procedures. Immunofluorescence was used to localize PLK4 and phospho-PLK4 in cultured human breast, ovarian, and colorectal cancer cell lines throughout the cell cycle without and with CFI-400945 PLK4 inhibitor and MG-115 protease inhibitor. Flow cytometry and videomicroscopy were used to analyze the consequences of PLK4 inhibition on cytokinesis. Results. Using immunofluorescence, PLK4 was localized to centrosomes; however, we also found that phospho-PLK4 was cleaved and distributed to kinetochores (metaphase and anaphase), cleavage furrow (telophase), and middle body (cytokinesis) during cell division in colorectal, ovarian, and breast cancer cells. Distribution of phospho-PLK4 to the cleavage furrow and middle body raised the possibility that this kinase plays a functional role in cytokinesis. Using either CFI-400945 PLK4 kinase inhibitor or the MG-115 protease inhibitor, we found that PLK4 accumulated in centrosomes with inhibition of translocation of PLK4 to the middle body. This change in subcellular distribution of PLK4 was associated with generation of large, multi-nucleated tumor cells or tumor cells with polyploidy. Videomicroscopy confirmed that treatment with CFI-400945 PLK4 inhibitor was associated with prevention of cellular abscission in treated cells. Conclusions. These observations demonstrate a role for phospho-PLK4 in facilitation of cytokinesis. A regulatory role for PLK4 in cytokinesis makes it a potential target for therapeutic intervention in appropriately selected cancers. Citation Format: Michael F. Press, Bin Xie, Simon Davenport, Yu Zhou, Neil O’Brien, Michael Palazzolo, Tak Mak, Joan Brugge, Dennis J. Slamon. Regulation of cytokinesis by polo-like kinase 4. [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 2736.