Joan T. Garrett

Transcriptional and posttranslational up-regulation of HER3 (ErbB3) compensates for inhibition of the HER2 tyrosine kinase

Sustained and complete inhibition of HER3 and its output to PI3K/Akt are required for the optimal antitumor effect of therapeutic inhibitors of the HER2 oncogene. Here, we show that, after inhibition of the HER2 tyrosine kinase with lapatinib, there is PI3K/Akt and FoxO3a-dependent up-regulation of HER3 mRNA and protein. Up-regulated HER3 was then phosphorylated by residual HER2 activity, thus partially maintaining P-Akt and limiting the antitumor action of lapatinib. Inhibition of HER3 with siRNA or a neutralizing HER3 antibody sensitized HER2+ breast cancer cells and xenografts to lapatinib both in vitro and in vivo. Combined blockade of HER2 and HER3 inhibited pharmacodynamic biomarkers of PI3K/Akt activity more effectively than each inhibitor alone. These results suggest that because of HER3-mediated compensation, current clinical inhibitors of HER2 and PI3K/Akt will not block the PI3K pathway completely. They also suggest that therapeutic inhibitors of HER3 should be used in combination with HER2 inhibitors and PI3K pathway inhibitors in patients with HER2- and PI3K-dependent cancers.

Mutations in the phosphatidylinositol 3-kinase pathway: role in tumor progression and therapeutic implications in breast cancer

Mutations in genes that constitute the phosphatidylinositol 3-kinase (PI3K) pathway occur in >70% of breast cancers. Clinical and experimental evidence suggest that PI3K pathway activation promotes resistance to some of the current breast cancer therapies. PI3K is a major signaling hub downstream of human epidermal growth factor receptor (HER)2 and other receptor tyrosine kinases. PI3K activates AKT, serum/glucocorticoid regulated kinase (SGK), phosphoinositide-dependent kinase 1 (PDK1), mammalian target of rapamycin (mTOR), and several other molecules involved in cell cycle progression and survival. In estrogen receptor (ER)+ breast cancer cells, PI3K activation promotes estrogen-dependent and -independent ER transcriptional activity, which, in turn, may contribute to anti-estrogen resistance. Activation of this pathway also confers resistance to HER2-targeted therapies. In experimental models of resistance to anti-estrogens and HER2 inhibitors, pharmacological inhibition of PI3K/AKT/mTOR has been shown to overcome drug resistance. Early clinical data suggest that combined inhibition of either HER2 or ER plus inhibition of the PI3K pathway might be an effective strategy for treatment of respective HER2+ and ER+ breast cancers resistant to standard therapies. Here, we review alterations in the PI3K pathway in breast cancer, their association with therapeutic resistance, and the state of clinical development of PI3K pathway inhibitors.

Resistance to HER2-directed antibodies and tyrosine kinase inhibitors: mechanisms and clinical implications.

The antibody trastuzumab and the tyrosine kinase inhibitor lapatinib are approved by the FDA for the treatment of HER2-overexpressing breast cancer. These anti-HER2 drugs are changing the natural history of HER2-overexpressing breast cancer. However, therapeutic resistance to trastuzumab or lapatinib, as either single-agents or in combination with chemotherapy in the metastatic setting, typically occurs within months of starting therapy. Several mechanisms of trastuzumab-resistance have been reported that include signaling from other HER receptors, signaling from receptor tyrosine kinases (RTKs) outside of the HER (ErbB) family, increased phosphatidylinositol 3-kinase signaling, and the presence of truncated forms of HER2. Mechanisms of resistance to lapatinib also point to increased phosphatidylinositol 3-kinase signaling as well as derepression/activation of compensatory survival pathways. In this review, we discuss how these models and mechanisms enhance our understanding of the clinical resistance to HER2-directed therapies.

An antibody that locks HER3 in the inactive conformation inhibits tumor growth driven by HER2 or neuregulin.

HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells, and it displayed single-agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations with similar activity in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope, within domains 2 and 4, that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that, in combination with HER2- or EGFR-targeted agents, may leverage their clinical efficacy in ErbB-driven cancers.

Dual blockade of HER2 in HER2-overexpressing tumor cells does not completely eliminate HER3 function

Purpose: Dual blockade of HER2 with trastuzumab and lapatinib or with pertuzumab is a superior treatment approach compared with single-agent HER2 inhibitors. However, many HER2-overexpressing breast cancers still escape from this combinatorial approach. Inhibition of HER2 and downstream phosphoinositide 3-kinase (PI3K)/AKT causes a transcriptional and posttranslational upregulation of HER3 which, in turn, counteracts the antitumor action of the HER2-directed therapies. We hypothesized that suppression of HER3 would synergize with dual blockade of HER2 in breast cancer cells sensitive and refractory to HER2 antagonists. Experimental Design: Inhibition of HER2/HER3 in HER2+ breast cancer cell lines was evaluated by Western blotting. We analyzed drug-induced apoptosis and two- and three-dimensional growth in vitro. Growth inhibition of PI3K was examined in vivo in xenografts treated with combinations of trastuzumab, lapatinib, and the HER3-neutralizing monoclonal antibody U3-1287. Results: Treatment with U3-1287 blocked the upregulation of total and phosphorylated HER3 that followed treatment with lapatinib and trastuzumab and, in turn, enhanced the antitumor action of the combination against trastuzumab-sensitive and -resistant cells. Mice bearing HER2+ xenografts treated with lapatinib, trastuzumab, and U3-1287 exhibited fewer recurrences and better survival than mice treated with lapatinib and trastuzumab. Conclusions: Dual blockade of HER2 with trastuzumab and lapatinib does not eliminate the compensatory upregulation of HER3. Therapeutic inhibitors of HER3 should be considered as part of multidrug combinations aimed at completely and rapidly disabling the HER2 network in HER2-overexpressing breast cancers. Clin Cancer Res; 19(3); 610–9. ©2012 AACR.

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.

ErbB3 Ablation Impairs PI3K/Akt-Dependent Mammary Tumorigenesis

The ErbB receptor family member ErbB3 has been implicated in breast cancer growth, but it has yet to be determined whether its disruption is therapeutically valuable. In a mouse model of mammary carcinoma driven by the polyomavirus middle T (PyVmT) oncogene, the ErbB2 tyrosine kinase inhibitor lapatinib reduced the activation of ErbB3 and Akt as well as tumor cell growth. In this phosphatidylinositol-3 kinase (PI3K)-dependent tumor model, ErbB2 is part of a complex containing PyVmT, p85 (PI3K), and ErbB3, that is disrupted by treatment with lapatinib. Thus, full engagement of PI3K/Akt by ErbB2 in this oncogene-induced mouse tumor model may involve its ability to dimerize with and phosphorylate ErbB3, which itself directly binds PI3K. In this article, we report that ErbB3 is critical for PI3K/Akt-driven tumor formation triggered by the PyVmT oncogene. Tissue-specific, Cre-mediated deletion of ErbB3 reduced Akt phosphorylation, primary tumor growth, and pulmonary metastasis. Furthermore, EZN-3920, a chemically stabilized antisense oligonucleotide that targets the ErbB3 mRNA in vivo, produced similar effects while causing no toxicity in the mouse model. Our findings offer further preclinical evidence that ErbB3 ablation may be therapeutically effective in tumors where ErbB3 engages PI3K/Akt signaling.

Novel Engineered Trastuzumab Conformational Epitopes Demonstrate In Vitro and In Vivo Antitumor Properties against HER-2/neu

Trastuzumab is a growth-inhibitory humanized Ab targeting the oncogenic protein HER-2/neu. Although trastuzumab is approved for treatment of advanced breast cancer, a number of concerns exist with passive immunotherapy. Treatment is expensive and has a limited duration of action, necessitating repeated administrations of the mAb. Active immunotherapy with conformational B cell epitopes affords the possibility of generating an enduring immune response, eliciting protein-reactive high-affinity anti-peptide Abs. The three-dimensional structure of human HER-2 in complex with trastuzumab reveals that the Ag-binding region of HER-2 spans residues 563–626 that comprises an extensive disulfide-bonding pattern. To delineate the binding region of HER-2, we have designed four synthetic peptides with different levels of conformational flexibility. Chimeric peptides incorporating the measles virus fusion “promiscuous” T cell epitope via a four-residue linker sequence were synthesized, purified, and characterized. All conformational peptides were recognized by trastuzumab and prevented the function of trastuzumab inhibiting tumor cell proliferation, with 563–598 and 597–626 showing greater reactivity. All epitopes were immunogenic in FVB/N mice with Abs against 597–626 and 613–626 recognizing HER-2. The 597–626 epitope was immunogenic in outbred rabbits eliciting Abs which recognized HER-2, competed with trastuzumab for the same epitope, inhibited proliferation of HER-2-expressing breast cancer cells in vitro and caused their Ab-dependent cell-mediated cytotoxicity. Moreover, immunization with the 597–626 epitope significantly reduced tumor burden in transgenic BALB-neuT mice. These results suggest the peptide B cell immunogen is appropriate as a vaccine for HER-2-overexpressing cancers because the resulting Abs show analogous biological properties to trastuzumab.

Peptide Vaccines of the HER-2/neu Dimerization Loop Are Effective in Inhibiting Mammary Tumor Growth In Vivo

Human epidermal growth factor receptor-2 (HER-2)/neu (ErbB2), a member of the epidermal growth factor family of receptors, is overexpressed in 20–30% of breast cancers. It is an attractive target for receptor-directed antitumor therapy using mAbs. Unlike other epidermal growth factor receptor family members, HER-2/neu does not bind a high-affinity ligand, but rather functions as the preferred dimerization partner. Pertuzumab (Omnitarg) is a humanized mAb directed against the HER-2/neu dimerization domain that inhibits receptor signaling. The recent definition of the crystal structure of the HER-2/neu-pertuzumab complex demonstrated that the receptor dimerization region encompassed residues 266–333. Based on the three-dimensional structure of the complex, we have designed three conformational peptide constructs (sequences 266–296, 298–333, and 315–333) to mimic regions of the dimerization loop of the receptor and to characterize their in vitro and in vivo antitumor efficacy. All the constructs elicited high-affinity peptide Abs that inhibited multiple signaling pathways including HER-2/neu-specific inhibition of cellular proliferation and cytoplasmic receptor domain phosphorylation. All the peptide Abs showed Ab-dependent cellular cytotoxicity to varying degrees with the 266–296 constructs being equally effective as compared with Herceptin. The 266–296 peptide vaccine had statistically reduced tumor onset in both transplantable tumor models (FVB/n and BALB/c) and significant reduction in tumor development in two transgenic mouse tumor models (BALB-neuT and VEGF+/−Neu2–5+/−). The 266–296 construct represents the most promising candidate for antitumor vaccination and could also be used to treat a variety of cancers with either normal or elevated expression of HER-2 including breast, lung, ovarian, and prostate.

LYN-activating mutations mediate antiestrogen resistance in estrogen receptor–positive breast cancer

Estrogen receptor-positive (ER(+)) breast cancers adapt to hormone deprivation and become resistant to antiestrogen therapy. Here, we performed deep sequencing on ER(+) tumors that remained highly proliferative after treatment with the aromatase inhibitor letrozole and identified a D189Y mutation in the inhibitory SH2 domain of the SRC family kinase (SFK) LYN. Evaluation of 463 breast tumors in The Cancer Genome Atlas revealed four LYN mutations, two of which affected the SH2 domain. In addition, LYN was upregulated in multiple ER(+) breast cancer lines resistant to long-term estrogen deprivation (LTED). An RNAi-based kinome screen revealed that LYN is required for growth of ER(+) LTED breast cancer cells. Kinase assays and immunoblot analyses of SRC substrates in transfected cells indicated that LYN(D189Y) has higher catalytic activity than WT protein. Further, LYN(D189Y) exhibited reduced phosphorylation at the inhibitory Y507 site compared with LYN(WT). Other SH2 domain LYN mutants, E159K and K209N, also exhibited higher catalytic activity and reduced inhibitory site phosphorylation. LYN(D189Y) overexpression abrogated growth inhibition by fulvestrant and/or the PI3K inhibitor BKM120 in 3 ER(+) breast cancer cell lines. The SFK inhibitor dasatinib enhanced the antitumor effect of BKM120 and fulvestrant against estrogen-deprived ER(+) xenografts but not LYN(D189Y)-expressing xenografts. These results suggest that LYN mutations mediate escape from antiestrogens in a subset of ER(+) breast cancers.