HER2 Kinase Domain Research Articles

S2-7: Mechanisms of Action and Biological Significance of HER2 Mutations in HER2−Overexpressing Breast Cancer.

Abstract Background: Trastuzumab is the most successful HER2−directed therapy in patients with early-stage and advanced HER2 positive breast cancer. Although trastuzumab improves survival in the adjuvant setting, 15–20% of the patients develop metastasis. Lapatinib is currently the only tyrosine kinase inhibitor approved for the treatment of patients with metastatic breast cancer after trastuzumab progression. However, progression eventually occurs and the disease remains incurable for the majority of patients. Recently, somatic mutations in the HER2 kinase domain have been reported in lung adenocarcinomas which result in constitutive phosphorylation of HER2, EGFR and cellular substrates. Activation of these receptors and pathways in lung cancer was associated with tumor progression and trastuzumab resistance, but tumors remained sensitive to a tyrosine kinase inhibitor. Because there are many different types of breast cancer that respond differently to treatments, more approaches are needed to predict which patients will most likely respond to a given therapy. Material and Methods: Using gene sequencing on 78 HER2 positive breast tumors, our laboratory identified 4 novel missense variants in the kinase domain of HER2. None of the patients whose tumors carried one of these mutations achieved objective response to trastuzumab. Mutants were created using directed mutagenesis, inserted in a lentiviral expression plasmid and stably expressed in different breast cancer or non-tumorigenic cell lines. An ATP-based assay was used for cell survival studies. Migration studies were performed using Boyden-chambers coated with Matrigel. Anchorage-independent colony formation was assessed in soft-agar. Signaling pathways and phosphorylation status were analyzed by Western blot. Localization results were obtained by immunofluorescence and confocal microscopy followed by deconvolution analysis. Results: Two of the mutants were dramatically under-phosphorylated and presented an altered cellular localization revealed by immunofluorescence studies in both cell lines and patient surgical samples. Also, cells expressing HER2 mutants showed an increased ability to invade Matrigel and migrate and to form colonies in soft-agar, suggesting the induction of a more aggressive behavior. In particular, one of the mutations was strongly associated with resistance to lapatinib treatment in cell survival and soft-agar assays, and higher doses of lapatinib were necessary to inhibit the ERK and AKT pathways. Computational analysis revealed that the mechanism of lapatinib resistance could be explained by a sterical obstruction of the ATP-binding pocket of the protein kinase domain that would impede the binding of lapatinib. Conclusion: HER2 mutations confer a more aggressive phenotype. A specific mutation directly interacts with Lapatinib binding to HER2 and predicts resistance to Lapatinib in HER2−overexpressing breast cancer cells. Further characterization of novel HER2 mutations may have a direct implication in the development of novel markers for early diagnostics, patient selection and characterization of more appropriate and personalized treatment. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S2-7.

Molecular and clinicopathologic characteristics of HER2-mutant lung adenocarcinoma (ADC).

10596 Background: Activating mutations within the tyrosine kinase domain of HER2 have been reported in a small subset of lung ADCs. HER2 mutations are mutually exclusive with EGFR and KRAS mutations and are associated with increased sensitivity to specific kinase inhibitors in preclinical models. To date, the frequency, molecular, and clinicopathologic characteristics of HER2 mutant lung ADC in the US have not been well defined. Methods: Consecutive clinical cases of lung ADC (n=419) known to be negative for mutations in EGFR (exons 19-21) and KRAS (codons 12-13) were selected based on DNA availability. HER2 mutation analysis was done by fragment analysis for small indels in exon 20 and a Sequenom assay panel for the most common point mutations. A subset of tumors (n=245) was tested for ALK fusions by FISH. Results: HER2 mutations were present in 4.8% (20/419) of lung ADCs. Nineteen (95%) were exon 20 insertions and 1 (5%) was a point mutation (L755S). None of the 16 HER2-mutants tested by FISH harbored an ALK fusion. The incidence of HER2 mutations in the “triple negative” (EGFR/KRAS/ALK) group was 6.5% (16/245). Morphologically, 75% (15/20) were moderately (9) or poorly differentiated (6) ADCs of mixed phenotype. Two (10%) were well differentiated and 3 could not be graded due to small sampling. Eight tumors had a bronchoalveolar component and 2 had a mucinous component. The patients (65% female) had a median age of 56 at diagnosis (range 43-87); 8 patients presented with advanced disease; 12 patients were never smokers. The prevalence of HER2 mutations was significantly higher (p=0.0005) in never smokers (13.5%) than in current/former smokers (2.6%) but there was no significant difference between females (5.8%) and males (4.9%). Conclusions: HER2 mutations in lung ADCs are mutually exclusive with EGFR and KRAS mutations and ALK fusions. The clinical and pathologic features of HER2 mutant patients show similarities to EGFR mutant cases. While the overall prevalence of these mutations is low, estimated at 2.2% of all lung ADCs, testing of “triple negative” patients identifies HER2 mutations in 6.5%, thereby more efficiently screening for this subset of patients who could potentially benefit from alternate targeted therapies.

Abstract PL2-1: Drug Development — Targeting HER2 beyond Trastuzumab

Abstract The use of trastuzumab in the treatment of early and advanced stage HER2 overexpressing (HER2+) breast cancers represents a significant advance in the treatment of breast cancer. In the advanced stage setting, the development of therapeutic resistance, either primary or secondary/acquired, is a clinical problem limiting the clinical efficacy of trastuzumab. This discussion will include alternative approaches to the treatment of HER2+ breast cancers, and strategies to enhance the clinical efficacy of trastuzumab by overcoming the development of therapeutic resistance. Elucidating mechanisms of resistance provides an opportunity to develop combination strategies based on scientific rationale to overcome or prevent its development. For example, the IGF1/IGF-1R signaling axis, which is frequently co-expressed in HER2+ breast cancers promotes trastuzumab resistance in preclinical models. IGF-1R inhibitors overcomes trastuzumab resistance in preclinical models, providing a strong rationale for evaluating the combination in clinical trials. Similarly, the pro-survival PI3K-Akt signaling network is frequently deregulated in breast cancers as a consequence of PI3KCA gain-of-function mutations or loss of the tumor suppressor PTEN. In preclinical models, deregulated PI3K signaling mediates therapeutic resistance to trastuzumab, and small molecule PI3K inhibitors have been shown to overcome trastuzumab resistance. Other mechanisms of resistance to trastuzumab include the presence of heregulin (HRG), a soluble growth factor ligand to HER3 and HER4, which promotes the formation of HER2/HER3 heterodimers, a complex that potently activates PI3K signaling. Other HER2 antibodies e.g. pertuzumab, are more effective at blocking the effects of HRG on HER2/HER3 dimerization. Another mechanism of resistance is truncation of HER2, resulting in a receptor that lacks the extracellular domain that contains the trastuzumab binding site. Strategies to prevent HER2 truncation have been examined preclinically. HER2 signaling has also been targeted using small molecules that compete with ATP for binding at the catalytic kinase domain of HER2. Lapatinib, an oral, reversible inhibitor of HER2 and EGFR tyrosine kinases is approved in combination with capecitabine for treating advanced stage HER2+ breast cancers that progressed on prior trastuzumab-based therapies. The dilemma is that the majority of women treated with lapatinib develop therapeutic resistance within 12 months of initiating therapy. Mechanisms of therapeutic resistance to lapatinib have been identified including (i) de-repression of estrogen receptor (ER) signaling, and (ii) activation of the pro-survival mediator NF-kB; the role of PTEN loss and the presence of PI3K activating mutations in the development of lapatinib resistance remains controversial. This insight has led to rational combinations to overcome or perhaps even delay the development of lapatinib resistance e.g. approval of letrozole plus lapatinib in HER2+/ER+ breast cancers. And finally, other HER2 targeted approaches will be discussed, including hsp90 antagonists, HER2 antibody conjugates (e.g. T-DM1), HER2 vaccines, and combinations of antibody-based and HER2 kinase inhibitors. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PL2-1.

Clinical Benefit of Lapatinib-Based Therapy in Patients with Human Epidermal Growth Factor Receptor 2–Positive Breast Tumors Coexpressing the Truncated p95HER2 Receptor

A subgroup of human epidermal growth factor receptor 2 (HER2)-overexpressing breast tumors coexpresses p95HER2, a truncated HER2 receptor that retains a highly functional HER2 kinase domain but lacks the extracellular domain and results in intrinsic trastuzumab resistance. We hypothesized that lapatinib, a HER2 tyrosine kinase inhibitor, would be active in these tumors. We have studied the correlation between p95HER2 expression and response to lapatinib, both in preclinical models and in the clinical setting. Two different p95HER2 animal models were used for preclinical studies. Expression of p95HER2 was analyzed in HER2-overexpressing breast primary tumors from a first-line lapatinib monotherapy study (EGF20009) and a second-line lapatinib in combination with capecitabine study (EGF100151). p95HER2 expression was correlated with overall response rate (complete + partial response), clinical benefit rate (complete response + partial response + stable disease > or =24 wk), and progression-free survival using logistic regression and Cox proportional hazard models. Lapatinib inhibited tumor growth and the HER2 downstream signaling of p95HER2-expressing tumors. A total of 68 and 156 tumors from studies EGF20009 and EGF100151 were evaluable, respectively, for p95HER2 detection. The percentage of p95HER2-positive patients was 20.5% in the EGF20009 study and 28.5% in the EGF100151 study. In both studies, there was no statistically significant difference in progression-free survival, clinical benefit rate, and overall response rate between p95HER2-positive and p95HER2-negative tumors. Lapatinib as a monotherapy or in combination with capecitabine seems to be equally effective in patients with p95HER2-positive and p95HER2-negative HER2-positive breast tumors.

Oncogenic mutations regulate tumor microenvironment through induction of growth factors and angiogenic mediators

Activating mutations in the tyrosine kinase domain of HER2 (ErbB2) have been identified in human cancers. Compared to wild-type HER2, mutant HER2 shows constitutively activate kinase activity and increased oncogenicity. Cells transformed by mutant HER2 are resistant to EGFR tyrosine kinase inhibitors and exhibit an attenuated response to the HER2 antibody trastuzumab. We investigated herein pathways through which mutant HER2 alters the extracellular environment, potentially leading to drug resistance and the effect of simultaneously targeting HER2 and the tumor cell microenvironment with a therapeutic intent. Expression of mutant HER2 in mammary epithelial cells activated autocrine transforming growth factor (TGF) β1 signaling through a mechanism involving Rac1 and JNK-AP1-dependent transcription. Cells transformed by an activating mutant of H-Ras (G12V) also expressed higher TGF-β1 level through Rac1 activation. In addition, mutant HER2 induced the EGFR ligands TGF-α and amphiregulin at the mRNA and protein levels. Vascular endothelial growth factor (VEGF), a target of the TGF-β-Smad transcriptional regulation, was also induced as a result of expression of mutant HER2. Inhibition of TGF-β signaling with the Alk5 small molecule inhibitor LY2109761 reduced growth and invasiveness of cells expressing mutant HER2. Combined inhibition of intracellular and paracrine effects of mutant HER2 by trastuzumab and the EGFR antibody cetuximab was more efficient than single-agent therapies. These data suggest that mutations in oncogenes such as HER2 and Ras not only alter intracellular signaling and also influence on other components of the tumor microenvironment by inducing several pro-invasive growth factors. In turn, these serve as extracellular targets of novel therapeutic strategies directed at both cancer-driving oncogenes and the modified tumor microenvironment.

Lapatinib is active in patients with HER2-amplified breast tumors expressing p95HER2.

Abstract Abstract #708 Background: A subgroup of HER2 overexpressing tumors also express p95HER2, a truncated fragment of the HER2 receptor that lacks the extracellular domain and retains kinase activity. Tumors expressing p95HER2 have a worse clinical outcome and are resistant to trastuzumab therapy (Scaltriti M. et al, JNCI 2007). The hypothesis that lapatinib, a small molecule inhibitor that targets the intracellular kinase domain of EGFR and HER2, would be active in these tumors was tested by correlating p95HER2 expression with clinical response to lapatinib in EGF20009, a lapatinib monotherapy study
 Methods: Archival tumor blocks were obtained from patients (pts) participating in study EGF20009. In this study, pts with untreated locally advanced or metastatic HER2 amplified breast tumors either received lapatinib 1500mg daily or 500mg BID (H.L. Gomez et al, J Clin Oncol 2008). Pre-treatment samples were analyzed for p95HER2 expression by immunofluorescence using 2 antibodies against HER2, binding to the intracellular and extracellular domain, respectively, plus a pan-cytokeratin antibody. Tumors were scored as p95HER2 positive (p95HER2+) [if any cytoplasmic staining was detected with the intracellular HER2 antibody and this staining co-localized with the pan-cytokeratin antibody], negative (p95HER2-) [no intracellular HER2 cytoplasmic staining) or non-evaluable (N/E) [no cytokeratin or HER2 staining by immunofluorescence]. The presence of p95HER2 was then correlated with response rate (RR), stable disease for over 100 days (SD) and progression-free survival (PFS) to lapatinib. A Cox-proportional hazards model was used to compare the groups. In order to correlate the presence of p95HER2 with HER2 extracellular domain in the serum, a quantitative determination of the 105 kD ECD level in serum (Oncogene Science HER2/neu Microtiter ELISA) has also been performed.
 Results: 68/105 tumors were evaluable for p95HER2 expression. Fourteen were p95HER2+ (20.5%) and 54 were p95HER2- (79.5%). The observed percentage of p95HER2+ tumors is concordant with published reports. Among the 14 pts with p95HER2+ tumors, 7 responded (RR: 50%) and 1 had SD for > 100 days for an overall clinical benefit rate of 57% (RR+SD). In the p95HER2- population (N=54), 24 pts responded (RR: 44.4%) and 16 pts had SD >100 days for an overall clinical benefit rate of 56% (RR+SD). PFS was similar in both groups, with a median PFS of 24 wks in pts with p95HER2+ and p95HER2- tumors (p=0.97, HR 1.1). These findings compare with the median PFS of 20.7 wks for the entire population in the trial (N=138pts). Further correlation of p95 status and HER2 ECD status of the tumors will be presented.
 Conclusion: These data demonstrate that lapatinib has similar activity in pts with p95HER2+ or p95HER2-, HER2 amplified breast tumors. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 708.

HER2 YVMA drives rapid development of adenosquamous lung tumors in mice that are sensitive to BIBW2992 and rapamycin combination therapy

Mutations in the HER2 kinase domain have been identified in human clinical lung cancer specimens. Here we demonstrate that inducible expression of the most common HER2 mutant (HER2(YVMA)) in mouse lung epithelium causes invasive adenosquamous carcinomas restricted to proximal and distal bronchioles. Continuous expression of HER2(YVMA) is essential for tumor maintenance, suggesting a key role for HER2 in lung adenosquamous tumorigenesis. Preclinical studies assessing the in vivo effect of erlotinib, trastuzumab, BIBW2992, and/or rapamycin on HER2(YVMA) transgenic mice or H1781 xenografts with documented tumor burden revealed that the combination of BIBW2992 and rapamycin is the most effective treatment paradigm causing significant tumor shrinkage. Immunohistochemical analysis of lung tumors treated with BIBW2992 and rapamycin combination revealed decreased phosphorylation levels for proteins in both upstream and downstream arms of MAPK and Akt/mTOR signaling axes, indicating inhibition of these pathways. Based on these findings, clinical testing of the BIBW2992/rapamycin combination in non-small cell lung cancer patients with tumors expressing HER2 mutations is warranted.

HER2 targeted therapy in breast cancer...beyond Herceptin

HER2, is a member of the ErbB family of receptor tyrosine kinases, that plays a key role in the pathogenesis of breast cancer, and when overexpressed, can correlate with a particularly aggressive clinical phenotype. It is estimated in roughly 18 to 20% of breast cancer patients, there is amplification of the HER2 gene resulting in aberrant overexpression of p185HER2 protein. As such, HER2 represents an attractive therapeutic target. In this review, we explore the clinical evolution of trastuzumab, a humanized monoclonal antibody with high specificity and affinity for the HER2 extracellular domain, and additionally examine more recent efforts targeting the intracellular tyrosine kinase domain of HER2 using small molecule tyrosine kinase inhibitors.

Surface Plasmon Resonance Investigations of Human Epidermal Growth Factor Receptor 2

This investigation utilizes surface plasmon resonance (SPR) spectroscopy to detect and quantify human epidermal growth factor receptor 2 (HER-2), an oncogene product that is over-expressed in some aggressive forms of breast cancer. Specifically, the HER-2 trans-membrane protein p185 and its extra cellular fragment p105 are analytes targeted in this work by using a gold-based biosensor slide on which an anti-HER-2 antibody has been immobilized by attachment to Protein G that is fixed to the gold film. A detection limit of > or =11 ng/mL for p185 resulted when trastuzumab was used as the anti-HER-2 antibody on the biosensor slide. Experiments with semi-purified p105 revealed that it binds weakly and reversibly to trastuzumab, therefore complicating its detection and quantification. Results of studies that reacted a 13-amino-acid peptide (PP13) from the HER-2 kinase domain with its specific antibody were critically different than p185 and p105 studies. Spectral analysis of the reflectivity at constant bulk buffer refractive index revealed a progressive negative SPR shift over time. A negative shift suggests that a loss of protein mass from the anti-PP13 antibody-Protein G biosensor is occurring. Several possibilities that may explain these negative SPR shifts are discussed.

HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors

HER2/Neu gene mutations have been identified in lung cancer. Expression of a HER2 mutant containing a G776(YVMA) insertion in exon 20 was more potent than wild-type HER2 in associating with and activating signal transducers, phosphorylating EGFR, and inducing survival, invasiveness, and tumorigenicity. HER2(YVMA) transphosphorylated kinase-dead EGFR(K721R) and EGFR(WT) in the presence of EGFR tyrosine kinase inhibitors (TKIs). Knockdown of mutant HER2 in H1781 lung cancer cells increased apoptosis and restored sensitivity to EGFR TKIs. The HER2 inhibitors lapatinib, trastuzumab, and CI-1033 inhibited growth of H1781 cells and cells expressing exogenous HER2(YVMA). These data suggest that (1) HER2(YVMA) activates cellular substrates more potently than HER2(WT); and (2) cancer cells expressing this mutation remain sensitive to HER2-targeted therapies but insensitive to EGFR TKIs.

Somatic Mutations of the HER2 Kinase Domain in Lung Adenocarcinomas

Somatic Mutations of the HER2 Kinase Domain in Lung Adenocarcinomas

Somatic Mutations of the HER2 Kinase Domain in Lung Adenocarcinomas

Mutations in the epidermal growth factor receptor gene (EGFR) in lung cancers predict for sensitivity to EGFR kinase inhibitors. HER2 (also known as NEU, EGFR2, or ERBB2) is a member of the EGFR family of receptor tyrosine kinases and plays important roles in the pathogenesis of certain human cancers, and mutations have recently been reported in lung cancers. We sequenced the tyrosine kinase domain of HER2 in 671 primary non-small cell lung cancers (NSCLC), 80 NSCLC cell lines, and 55 SCLCs and other neuroendocrine lung tumors as well as 85 other epithelial cancers (breast, bladder, prostate, and colorectal cancers) and compared the mutational status with clinicopathologic features and the presence of EGFR or KRAS mutations. HER2 mutations were present in 1.6% (11 of 671) of NSCLC and were absent in other types of cancers. Only one adenocarcinoma cell line (NCI-H1781) had a mutation. All HER2 mutations were in-frame insertions in exon 20 and target the identical corresponding region as did EGFR insertions. HER2 mutations were significantly more frequent in never smokers (3.2%, 8 of 248; P=0.02) and adenocarcinoma histology (2.8%, 11 of 394; P=0.003). In 394 adenocarcinoma cases, HER2 mutations preferentially targeted Oriental ethnicity (3.9%) compared with other ethnicities (0.7%), female gender (3.6%) compared with male gender (1.9%) and never smokers (4.1%) compared with smokers (1.4%). Mutations in EGFR, HER2, and KRAS genes were never present together in individual tumors and cell lines. The remarkable similarities of mutations in EGFR and HER2 genes involving tumor type and subtype, mutation type, gene location, and specific patient subpopulations targeted are unprecedented and suggest similar etiologic factors. EGFR, HER2, and KRAS mutations are mutually exclusive, suggesting different pathways to lung cancer in smokers and never smokers.

Tyrphostins. 3. Structure-activity relationship studies of alpha-substituted benzylidenemalononitrile 5-S-aryltyrphostins.

In this study we describe an extension of our previous studies on cis-benzylidenemalononitrile tyrphostins. We have introduced S-aryl substituents in the 5 position (meta vis-a-vis the malononitrile moiety). We find that these compounds are potent blockers of EGFR kinase and its homolog HER-2 kinase. Interestingly, we find that certain S-aryltryphostins discriminate between EGFR and HER-2 kinase in favor of the HER-2 kinase domain by almost 2 orders of magnitude. When examined in intact cells it was found that these selective S-aryltrphostins are equipotent in inhibiting EGF dependent proliferation of NIH 3T3 harboring either the EGF receptor or the chimera EGF/neu (HER1-2). These findings suggest that the antiproliferative activity of these tyrphostins is mainly due to the inhibition of a mitogenic signaling element downstream to the growth receptor kinase.

Selective inhibition of the epidermal growth factor and HER2/neu receptors by tyrphostins

The HER2 (neu/erb-B2) proto-oncogene codes for a transmembrane receptor with tyrosine kinase activity and with high homology to the EGF receptor (HER1). The high incidence of HER2 overexpression in breast and ovary carcinomas prompted us to synthesize protein tyrosine kinase inhibitors (tyrphostins) which selectively inhibit the HER2 kinase activity. Two groups of tyrphostins were developed: one highly selective in inhibiting HER1 as opposed to HER2, the other highly selective in inhibiting HER2. Both the HER1 and the HER2 selective blockers were competitive with ATP binding. This suggests that even though the kinase domains of the respective receptors show an 80% degree of homology it is possible to design small molecules capable of discriminating between them. These results also show that the two kinases differ in their ATP binding sites. Mitogenic signaling induced by EGF in NIH3T3 cells overexpressing either HER1 or HER1-2 (possessing the HER2 kinase domain) was blocked identically by the agents that discriminate between the two in vitro. This paradox was further explored and elucidated. We propose that high intracellular ATP levels prevent inhibitor binding to the receptor. The antiproliferative action of the two distinct selective tyrphostins observed may result from the inhibition of a downstream element, presumably a tyrosine kinase, which mediates mitogenic signaling.