Abstract NG01: Blockade of EGFR and HER3 enhances PI3K/Akt antitumor activity in triple negative breast cancer
Authors: Scaltriti Maurizio , Tao Jessica , Castel Pau , Radosevic-Robin Nina , Elkabets Moshe , Auricchio Neil , Juric Dejan , Penault-Llorca Frédérique , Baselga Jose 
Published: May 2015

Abstract
Triple negative breast cancer (TNBC) is clinically defined by lack of expression of estrogen and progesterone receptors and no overexpression/amplification of human epidermal growth factor receptor 2 (HER2). It affects approximately one-fourth of breast cancer patients and is usually characterized by an aggressive phenotype with high mitotic index and poor prognosis. No TNBC-targeted therapeutic exists and the standard of care is currently limited to cytotoxic chemotherapy.
Overexpression of human epidermal growth factor receptor (EGFR) as well as inactivation or decreased expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) are frequent in TNBC. Thus, inhibition of EGFR and/or the PI3K/Akt pathway are attractive therapeutic strategies for this malignancy. Preclinical evidence, however, suggests that inhibition of the PI3K/Akt axis induces compensatory expression and activation of upstream receptor tyrosine kinases, including EGFR and, most prominently, human epidermal growth factor receptor 3 (HER3). This may reduce the antitumor effects of single-agent PI3K pathway blockade. Furthermore, studies using in vitro models of resistance to the anti-EGFR antibody cetuximab suggest that HER3 expression itself can limit the sensitivity to this agent by increasing EGFR/HER3 heterodimerization and activation of downstream pathways.
In this work we hypothesized that targeting both EGFR and HER3, in combination with PI3K/Akt inhibition, would result in enhanced therapeutic activity in EGFR-positive TNBC.
We treated PTEN-negative EGFR-positive HCC70 and MDA-MB-468 TNBC cell lines with GDC-0068, a selective inhibitor of the Akt 1/2/3 isoforms, GDC-0941, a class I selective pan-PI3K inhibitor, MEHD7945A, an antibody targeting both EGFR and HER3 and the combinations of these inhibitors. Compared to single agents, the combination of GDC-0068 or GDC-0941 and MEHD7945A prevented the induction of EGFR and HER3 phosphorylation, enhanced the inhibition of the PI3K and ERK downstream pathways and resulted in superior inhibition of cell proliferation/viability compared to single agents.
To expand our findings in vivo, we first tested the efficacy of MEHD7945A in combination with either the Akt or PI3K inhibitor in both MDA-MB-468- and HCC70-derived xenografts. While the tumors responded only modestly to single agent GDC-0068, GDC-0941 and MEHD7945A, the combination of GDC-0068 or GDC-0941 with MEHD7945A yielded superior tumor growth inhibition compared to monotherapies. We next investigated the levels of EGFR and HER3 expression/activation in HCC70 tumors collected at the end of the experiments by reverse-phase protein detection. As expected, increase of both EGFR and HER3 expression and phosphorylation induced by Akt or PI3K was prevented by the addition of MEHD7945A.
We then tested the activity of the same treatments in a patient-derived xenograft (PDX) model of TNBC available in our laboratory. These tumors were characterized by undetectable levels of PTEN, high levels of EGFR and ∼70% staining for Ki67. When used as single agents, GDC-0068, GDC-0941 and MEHD7945A delayed tumor growth. However, their combinations elicited durable tumor stasis. Consistently with the cell-based xenografts, MEHD7945A counteracted EGFR and HER3 phosphorylation caused by PI3K/Akt inhibition. Collectively, these data show that targeting both EGFR and HER3 enhances the antitumor effects of PI3K/Akt inhibitors.
In order to dissect the role of HER3 inhibition in these models, we compared the activity of cetuximab with MEHD7945A in combination with either GDC-0068 or GDC-0941 in HCC70 cells. Both antibodies enhanced the antiproliferative activity mediated by PI3K/Akt inhibition in cells stimulated with the EGFR ligand epidermal growth factor; however MEHD7945A was superior to cetuximab in cooperating with the antiproliferative activity of GDC-0068 and GDC-0941 in cells stimulated with the HER3 ligand heregulin. We next compared the antitumor activity of cetuximab and MEHD7945A in combination with GDC-0941 in HCC70-derived xenografts. While concomitant targeting of EGFR, HER3 and PI3K was superior to monotherapies and led to tumor shrinkage, the combination of cetuximab and GDC-0941 did not result to any further inhibition of tumor growth compared to single agent treatments. Biochemically, both MEHD7945A and cetuximab blocked EGFR phosphorylation in these xenografts; however, only MEHD7945A decreased HER3 activation. These results confirm that HER3 plays an important role in limiting the efficacy of PI3K inhibition in this setting.
To investigate whether changes in EGFR and HER3 levels can affect the response to anti-EGFR therapy in TNBC patients, we measured the expression of these receptors in samples from patients enrolled in two pilot neoadjuvant clinical trials testing the antitumor activity of the anti-EGFR antibodies panitumumab (40 patients) and cetuximab (30 patients) in combination with standard chemotherapy. Of 40 patients treated with panitumumab-based therapy 19 (47.5%) achieved pathological complete response (pCR) after 24 weeks and 21 (52.5%) showed residual disease at the time of surgery. We measured EGFR and HER3 expression by immunohistochemistry in the 40 pre-treatment specimens and in the 21 residual invasive tumors excised at surgery. Patients with higher baseline EGFR expression were more likely to achieve pCR. Moreover, EGFR levels were decreased in the residual tumors of nine out of 21 non-pCR patients when compared to the paired baseline specimens (p=0.07). HER3 staining, available for 13 non-responder patients, showed higher HER3 expression in the residual lesions of 7 out of 13 non-pCR patients compared to the paired pre-treatment samples (p=0.01).
Of 30 patients enrolled in the study testing the antitumor activity of cetuximab-based therapy, 9 experienced pCR. Consistently, HER3 expression was found upregulated in the residual tumors of 11 out of 19 non-pCR patients when compared to the baseline paired specimens (p=0.103).
These results suggest that high EGFR expression may be required for optimal response to anti-EGFR therapeutic antibodies and that HER3 expression increases following anti-EGFR therapy in patients that do not experience complete tumor regression.
In this work we provide evidence that HER3 plays a potential role in limiting the antitumor activity of both PI3K/Akt inhibitors and anti-EGFR agents. We demonstrate that simultaneous targeting of EGFR and HER3 is required to enhance the efficacy of PI3K/Akt inhibition in preclinical models of EGFR-positive TNBC. Further, our clinical findings suggest that HER3 expression is induced in TNBC patients with a lower probability of achieving tumor remission upon anti-EGFR therapy.
Therefore, the rationale behind targeting simultaneously EGFR, HER3 and PI3K in TNBC is based on: 1) nearly half of TNBCs express high levels of EGFR, 2) TNBCs often express low levels of PTEN and 3) HER3 upregulation may limit their sensitivity to targeted therapy. In addition, tumor cells that express relatively high levels of these receptors can function as molecular “flags” for immune-mediated antibody dependent cytotoxicity, intrinsic characteristics of IgG type I antibodies such as MEHD7945A in vivo.
Given that both pharmacokinetic and pharmacodynamic data for MEHD7945A, GDC-0068 and GDC-0941 are already available, the design of phase II clinical trials testing the activity of these possible combinations in TNBC would be straightforward. Patient enrollment may be based on high EGFR expression with the requirement of mandatory “on-treatment” biopsies to evaluate both pathway inhibition and possible HER3 upregulation upon therapy.
In conclusion, we believe that simultaneous inhibition of EGFR, HER3 and the PI3K/Akt pathway has the potential to greatly expand the percentage of TNBC patients who can benefit from targeted therapy.
Citation Format: Maurizio Scaltriti, Jessica Tao, Pau Castel, Nina Radosevic-Robin, Moshe Elkabets, Neil Auricchio, Dejan Juric, Frédérique Penault-Llorca, Jose Baselga. Blockade of EGFR and HER3 enhances PI3K/Akt antitumor activity in triple negative 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 NG01. doi:10.1158/1538-7445.AM2014-NG01