HER2-amplified Cancer Cells Research Articles

Abstract P234: ORIC-114, an orally bioavailable, irreversible kinase inhibitor, has superior brain penetrant properties and enhanced potency in preclinical studies of HER2-positive breast cancer

Abstract Amplification of human epidermal growth factor receptor 2 (HER2) is an oncogenic driver found in approximately 25% of breast cancer. Despite the arsenal of HER2-directed therapies available to patients, more than 50% of patients with HER2 amplification eventually develop central nervous system (CNS) metastases over the course of their disease indicating a clear medical need for brain penetrant therapies in this patient population. ORIC-114 is a brain penetrant, orally bioavailable, irreversible small molecule inhibitor that was designed to target exon20 insertions in epidermal growth factor receptor (EGFR) and HER2. ORIC-114 is highly selective for the EGFR/HER2 family of receptors, reducing the risk for off-target kinase liabilities. In biochemical assays, ORIC-114 displayed low nanomolar potency on HER2. To explore the application of ORIC-114 in the HER2-amplified tumor setting, a cell viability screen was performed against a panel of human breast cancer lines containing both HER2-amplified and non HER2-amplified cell lines. ORIC-114 demonstrated greater than 100-fold enhanced cellular potency on HER2-amplified cancer cell lines relative to non-amplified cancer cell lines. Notably, ORIC-114 cellular EC50s in HER2-amplified breast cancer cell lines were below 30 nM and more potent than both lapatinib and tucatinib, two FDA-approved tyrosine kinase inhibitors for the treatment of HER2-positive breast cancer. ORIC-114 was designed to incorporate physicochemical properties suitable to cross the blood-brain barrier and has exhibited good brain penetration across multiple preclinical species. To further investigate the brain penetrant attributes of ORIC-114 in the context of HER2-positive breast cancer with brain metastases, key features were assessed relative to tucatinib, which has demonstrated clinical efficacy in this setting. In contrast to tucatinib, ORIC-114 displayed minimal impact on efflux transporters as it was only a weak substrate for P-glycoprotein (P-gp) and was not a substrate for breast cancer associated protein (BCRP) in vitro. In addition, ORIC-114 demonstrated superior in vivo brain penetration relative to tucatinib as measured by free brain-to-plasma ratio in mouse. Consequently, ORIC-114 free brain concentrations in rodents were greater than tucatinib, even when the active metabolites of tucatinib were considered. Taken together, these data further establish ORIC-114 as a selective, irreversible, and brain penetrant EGFR/HER2 inhibitor, making it a promising therapeutic candidate for development in patients with HER2-positive tumors including those with CNS metastases. A Clinical Trial Application for ORIC-114 is anticipated in the second half of 2021. Citation Format: Melissa R. Junttila, Jason E. Long, Robert Warne, Sunghwan Kim, Younho Lee, Hwan Kim, Juhee Kang, Jiyoon Seok, Jihye Yoo, Youngyi Lee, Dong-Hyuk Seo Seo, Jung Beom Son, Daekwon Kim, Hwan Geun Choi, Nam Doo Kim, Tatiana Zavorotinskaya, Chelsea Chan, Matthew Panuwat, Jessica Sun, Jae H. Chang, Lori S. Friedman. ORIC-114, an orally bioavailable, irreversible kinase inhibitor, has superior brain penetrant properties and enhanced potency in preclinical studies of HER2-positive breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P234.

The role of HER2 and HER3 in HER2-amplified cancers beyond breast cancers

HER2 and HER3 play key driving functions in the pathophysiology of HER2-amplified breast cancers, but this function is less well characterized in other cancers driven by HER2 amplification. This study aimed to explore the role of HER2 and HER3 signaling in other types of HER2-amplified cancer. The expression and signaling activity of HER2, HER3, and downstream pathway proteins were studied in cell panels representing HER2-amplified cancers of the breast, bladder, colon and rectal, stomach, esophagus, lung, tongue, and endometrium along with controls lacking HER2 amplification. We report that HER2-amplified cancers are addicted to HER2 across different cancer types and the depth of addiction is best linked with the expression level of HER2, but not with HER3 expression. We report that the expression and constitutive phosphorylation of HER3 are ubiquitous in HER2-amplified breast cancer cell lines, but much more variable in HER2-amplified cancer cells from other tissues. We observed the lapatinib-induced compensatory upregulation of HER3 signaling in many types of HER2-amplified cancers, although with much variability. We find that HER3 expression is essential for in vivo tumorigenic growth in some HER2-amplified tumors but not others. Importantly HER3 expression level does not correlate well with its functional importance. More biomarkers will be needed to guide the optimal use of HER3 inhibitors in HER2-amplified cancers from non-breast origin. Unlike oncogenes activated through mutational events, the activation of HER2 through overexpression represents a gradient of activities and depth of addiction and the response to inhibitors follows a similar gradient.

Abstract LB-326: The role of HER3 in HER2-amplified cancers other than breast cancers

Abstract Introduction: Although recent studies showed that HER3 plays a key role in the pathophysiology of HER2-amplified breast cancers, its role in other cancers driven by HER2 amplification remains unknown. This study aimed to define the importance of HER3 signaling in other types of HER2-amplified cancer. Method: The expression and signaling activity of HER2, HER3, and downstream pathway proteins were studied in cell panels representing HER2-amplified cancers of the breast, bladder, colon and rectal, stomach, esophagus, lung, tongue, and endometrium along with controls lacking HER2 amplification. The time-dependent compensatory upregulation of HER3 in response to HER2 inhibitor therapy was assayed by western blotting and the apoptotic effects of HER2 inhibition were assayed by flow cytometry. The functional significance of HER3 in promoting tumorigenic growth in xenograft assays was determined in selected HER2-amplified cancer cell types by shRNA knockdown. Results: Many HER2-amplified cancers of the breast (HCC1569, HCC1954, HCC1419, UACC732, SUM190), bladder (CLS-439), stomach (N87, OE19), esophagus (TE4, KYSE410), lung (Calu3), and endometrium (USPC-ARC1, USPC-ARC2) showed HER3 activation, whereas HER2-nonamplified cancers of the same organs did not. With some exceptions, all HER2-amplified cancer cells undergo apoptosis when HER2 signaling is inhibited by lapatinib. Lapatinib induced apoptosis correlated with HER3 activation. Tumor growth was reduced upon HER3 knockdown in HER2-amplified cancers other than breast cancers, particularly cancers with constitutive HER3 phosphorylation. Significance: These results suggest that HER3 plays a functionally significant role in HER2 amplified cancers other than breast cancers, and may underlie resistance to HER2-targeting in these cancers. Combined HER2-HER3 targeting may be of value across the spectrum of different types of cancers. Citation Format: Avisek Majumder, Veronica Steri, Fernando Salangsang, Mark Moasser. The role of HER3 in HER2-amplified cancers other than breast cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-326.

Abstract B66: The conditional requirement of HER3 in HER2-amplified breast cancers

Abstract The amplification and overexpression of HER2 underlies the pathogenesis of a large subset of breast, gastric, and esophageal cancers as well as smaller subsets of many other types of cancer. A substantial body of work has confirmed that overactive HER2 signaling is etiologically linked with the pathogenesis, progression and metastasis of these cancers. HER3, the preferred heterodimerization partner of HER2, is known to be critical for HER2-amplified tumorigenesis, and this has been thought to be due to its unique ability to activate PI3K/Akt pathway. This has fueled efforts to develop HER3 targeting agents to combine with HER2 inhibitors for the treatments of these cancers, and many such agents are in the preclinical and clinical pipelines. We have been modeling HER2-HER3 signaling co-targeting in experimental models to foresee the activities and limitations of this clinical strategy and have identified potentially significant escape mechanisms that limit the therapeutic potential of this approach. This appears to be largely due to the mistaken attribution of PI3K/Akt signaling to HER3 and the dogma that HER2 cannot activate PI3K by itself. The functions of HER3 are not entirely understood and may be partly redundant with HER2, enabling HER2 to escape the requirement for HER3. In this regard, we found that HER3-knock out HER2-overexpressing cells form tumors, and some of these tumors exhibit increased HER2 phosphorylation and Akt activation. We validated, in three different HER2-amplified cancer cell lines, that phosphorylated HER2 drives PI3K-mediated Akt activation in a HER3-independent manner. Interestingly, we found that HER2 interacts with the regulatory subunit of PI3K in these cells. We characterized this interaction and identified a tyrosine in the C-terminal tail of HER2 involved in PI3K binding. Most importantly, in vivo work demonstrates that mutation of this tyrosine reduces the transforming and tumorigenic potential of HER2-amplified cancer cells. This work highlights that the activation of PI3K/Akt signaling by HER3 may be an important but not essential function and may ultimately be redundant with the functional capabilities of massively overexpressed HER2. Therefore, building a better understanding of the molecular mechanisms that underlie HER2-HER3-induced tumorigenesis is essential for the development of more effective therapeutic strategies. Citation Format: Ana Ruiz-Saenz, Courtney Dreyer, Veronica Steri, Marcia Campbell, Nathaniel Gulizia, Mark Moasser. The conditional requirement of HER3 in HER2-amplified breast cancers [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B66.

HER2 Amplification in Tumors Activates PI3K/Akt Signaling Independent of HER3.

Current evidence suggests that HER2-driven tumorigenesis requires HER3. This is likely due to the unique ability of HER3 to activate PI3K/Akt pathway signaling, which is not directly accessible to HER2. By genetic elimination of HER3 or shRNA knockdown of HER3 in HER2-amplified cancer cells, we find residual HER2-driven activation of PI3K/Akt pathway signaling that is driven by HER2 through direct and indirect mechanisms. Indirect mechanisms involved second messenger pathways, including Ras or Grb2. Direct binding of HER2 to PI3K occurred through p-Tyr1139, which has a weak affinity for PI3K but becomes significant at very high expression and phosphorylation. Mutation of Y1139 impaired the tumorigenic competency of HER2. Total elimination of HER3 expression in HCC1569 HER2-amplified cancer cells significantly impaired tumorigenicity only transiently, overcome by subsequent increases in HER2 expression and phosphorylation with binding and activation of PI3K. In contrast to activation of oncogenes by mutation, activation by overexpression was quantitative in nature: weak intrinsic activities were strengthened by overexpression, with additional gains observed through further increases in expression. Collectively, these data show that progressive functional gains by HER2 can increase its repertoire of activities such as the activation of PI3K and overcome its dependency on HER3.Significance: The intrinsic ability of HER2 to activate PI3K correlates with increased HER2 expression and can supplant the dependency upon HER3 for growth in HER2-amplified cancers. Cancer Res; 78(13); 3645-58. ©2018 AACR.

PO-118 The expression of the calcium-sensing receptor in colon organoids

IntroductionAmplification and overexpression of HER2 underlies the pathogenesis of a large subset of cancers including more than 20% of breast cancers. A considerable body of experimental evidence strongly suggests that HER2 is the oncogenic driver of these cancers, important in the genesis and progression of these tumours. Current evidence suggests that HER2 driven tumorigenesis requires HER3, the preferred heterodimerization partner of HER2. This is felt to be due to the unique ability of HER3 to activate PI3K/Akt pathway signalling, not directly accessible to HER2. We have been interested in developing deeper insights into the role of HER3 in HER2-amplified cancers. In particular, we have been interested in understanding why HER3 is required, whether the requirement is for the activation of PI3K, and whether its requirement is absolute or perhaps conditional or transient.Material and methodsUsing CRISPR-Cas9 technology, we knocked-out HER3 (HER3KO) in HER2-amplified HCC1569 cells and assessed the transforming and tumorigenic properties of HER3KO cells in vitro and in vivo. Co-immunoprecipitation assays in HER3-KO cells and stable doxycycline-inducible HER3 shRNA cell lines were performed to detect the binding of HER2 to the regulatory subunit of PI3K through direct and indirect mechanisms. Site-directed mutagenesis in the C-terminal of HER2 was used to identify the tyrosine involved in this interaction.Results and discussionsBy genetic elimination of HER3 or shRNA knockdown of HER3 in HER2-amplified cancer cells we find residual HER2-driven activation of PI3K/Akt pathway signalling that is driven by HER2 through direct and indirect mechanisms. Indirect mechanisms involve second messenger pathways including Ras or Grb2. Direct PI3K binding occurs through p-Tyr1139 which has a weak affinity for PI3K that becomes significant at very high expression and phosphorylation. Mutation of Y1139 impairs the tumorigenic competency of HER2. The total elimination of HER3 expression in HER2-amplified cancer cells significantly impairs tumorigenicity, but the impairment is transient and overcome with further increases in HER2 expression and phosphorylation with binding and activation of PI3K. Effective treatment of these cancers must focus on much more potent inhibitors of HER2.ConclusionThis study shows for first time the intrinsic ability of HER2 to activate PI3K that becomes more relevant with increased HER2 expression, and can supplant the requirement for HER3 in the growth of HER2-amplified cancers.

PO-115 HER2-amplified tumours overcome the requirement for HER3

ABSTRACT Introduction Amplification and overexpression of HER2 underlies the pathogenesis of a large subset of cancers including more than 20% of breast cancers. A considerable body of experimental evidence strongly suggests that HER2 is the oncogenic driver of these cancers, important in the genesis and progression of these tumours. Current evidence suggests that HER2 driven tumorigenesis requires HER3, the preferred heterodimerization partner of HER2. This is felt to be due to the unique ability of HER3 to activate PI3K/Akt pathway signalling, not directly accessible to HER2. We have been interested in developing deeper insights into the role of HER3 in HER2-amplified cancers. In particular, we have been interested in understanding why HER3 is required, whether the requirement is for the activation of PI3K, and whether its requirement is absolute or perhaps conditional or transient. Material and methods Using CRISPR-Cas9 technology, we knocked-out HER3 (HER3KO) in HER2-amplified HCC1569 cells and assessed the transforming and tumorigenic properties of HER3KO cells in vitro and in vivo. Co-immunoprecipitation assays in HER3-KO cells and stable doxycycline-inducible HER3 shRNA cell lines were performed to detect the binding of HER2 to the regulatory subunit of PI3K through direct and indirect mechanisms. Site-directed mutagenesis in the C-terminal of HER2 was used to identify the tyrosine involved in this interaction. Results and discussions By genetic elimination of HER3 or shRNA knockdown of HER3 in HER2-amplified cancer cells we find residual HER2-driven activation of PI3K/Akt pathway signalling that is driven by HER2 through direct and indirect mechanisms. Indirect mechanisms involve second messenger pathways including Ras or Grb2. Direct PI3K binding occurs through p-Tyr1139 which has a weak affinity for PI3K that becomes significant at very high expression and phosphorylation. Mutation of Y1139 impairs the tumorigenic competency of HER2. The total elimination of HER3 expression in HER2-amplified cancer cells significantly impairs tumorigenicity, but the impairment is transient and overcome with further increases in HER2 expression and phosphorylation with binding and activation of PI3K. Effective treatment of these cancers must focus on much more potent inhibitors of HER2. Conclusion This study shows for first time the intrinsic ability of HER2 to activate PI3K that becomes more relevant with increased HER2 expression, and can supplant the requirement for HER3 in the growth of HER2-amplified cancers.

The novel dithiocarbamate, DpdtC suppresses HER2-overexpressed cancer cells by up-regulating NDRG1 via inactivation of HER2-ERK 1/2 signaling

Dithiocarbamate has been tested for its effective anti-tumor activity, but the underlying mechanism remains unclear. We previously prepared a novel diththiocarbamate derivative, DpdtC with an ability of catalase inhibition. Here, we for the first time investigated the growth inhibition effects of DpdtC on HER2-amplified cancer cells and elucidated its mechanism of action. Results showed that DpdtC exerted the potent anti-tumor effects against HER2-overexpressed SK-OV-3 and SK-BR-3 cells, especially on SK-OV-3 cells with a higher NDRG1 level, which was also confirmed in the SK-OV-3 xenograft model. Interestingly, we observed that NDRG1 was up-regulated, while membrane expression of HER2 was regressed in SK-OV-3 cells upon DpdtC treatment. In agreement, silencing endogenous NDRG1 also increased the expression of HER2 in SK-OV-3 cells, while overexpressing NDRG1 decreased HER2 expression in SK-BR-3 cells. Furthermore, our results showed the formation of the EGFR/HER2 heterodimer was attenuated and phosphorylation of ERK1/2 was inhibited in SK-OV-3 cells when treated with DpdtC. Collectively, these observations demonstrated that NDRG1 plays an important role in mediating the inhibition effects of DpdtC in HER2-overexpressed cancer cells via selective targeting of the HER2-ERK1/2 pathway. Hence, our investigation suggests that up-regulation of NDRG1 by DpdtC is a promising therapeutic approach in HER2-overexpressed cancers.

Chemical probing of HER2-amplified cancer cells identifies TORC2 as a particularly effective secondary target for combination with lapatinib.

The clinical impact of HER2 inhibitors in the treatment of HER2-amplified breast cancers has been largely confined to chemotherapy combination regimens, since HER2 inhibitors appear to have very modest efficacies by themselves. This is due to the resilient nature of the functionally relevant HER2-HER3 tumor driver, bidirectionally linked with downstream PI3K/Akt pathway signaling, which can break through the inhibitory effects of most current HER2 or HER3 targeting therapies. A vertical combination approach targeting HER2 and a downstream pathway is a highly rational strategy for much more effective targeted therapy of this disease. However the importance of these downstream pathways in many human tissues and cells significant limits their usefulness as secondary targets by narrowing the therapeutic index of such combination therapies. The secondary target that can afford the highest potential for clinical translation is the one with the highest synergy against tumor cells in combination with HER2-inhibition, allowing the widest therapeutic index for clinical translation. We conducted a comparative analysis of such secondary targets in combination with the HER2 inhibitor lapatinib and find that the inhibition of mTor affords the highest degree of synergy. In further dissecting the individual roles of TORC1 and TORC2 complexes using pharmacologic and genetic tools, we find that it is specifically the inactivation of TORC2 that most synergistically enhances the efficacy of lapatinib. Although inhibitors that selectively target TORC2 are not currently available, these data make a compelling case for their development.

Abstract LB-261: Mechanism of action of MCLA-128, a humanized bispecific IgG1 antibody targeting the HER2:HER3 heterodimer

Abstract Background: MCLA-128 is an ADCC-enhanced humanized common light chain bispecific IgG1 antibody that targets the HER2:HER3 dimer with nanomolar affinity, potently inhibiting tumor growth in vitro and in vivo. MCLA-128 shows superior activity to the combination trastuzumab/ pertuzumab and HER3 targeting monoclonal antibodies and is currently being evaluated in a Phase I clinical trial. This study investigated the mechanism of action of MCLA-128. Methods: Phosphorylation of HER receptors and downstream signaling molecules was studied in vitro and in vivo on HER2-amplified cancer cell lines by Pathscan arrays, luminex beads and Western blot analysis. Inhibition of MCLA-128 cell growth in combination with tyrosine kinase inhibitors and small molecules targeting the MAPK and PI3 kinase/Akt pathway was determined by proliferation inhibition and high content imaging assays. The potential effect of MCLA-128 on primary cardiomyocytes in the presence of Doxorubicin was analyzed by measuring ATP. Binding of MCLA-128 to a panel of cell lines in comparison to HER2 and HER3 antibodies was determined by FACS. Results: In contrast to other HER2 and HER3 targeted agents, only MCLA-128 inhibited phosphorylation of HER3 and downstream Akt and ERK in HER2 amplified cell lines cultured with high concentrations of heregulin in vitro. In xenograft studies, growth inhibition of the trastuzumab-resistant cell line JIMT-1 by MCLA-128 was correlated with reduced HER2:HER3 dimerization and a profound inhibition of the PI3K pathway. Synergistic growth inhibition in vitro was observed when tyrosine kinase inhibitors or inhibitors of the PI3K pathway were added to HER2 amplified cancer cells in the presence of MCLA-128. MCLA-128 did not show any evidence of cardiotoxicity in vitro in contrast to trastuzumab. MCLA-128 binds and coats breast cancer cell lines with differing levels of HER2 expression more efficiently in comparison to monospecific HER2 or HER3 monoclonal antibodies. Conclusions: The unique simultaneous targeting of MCLA-128 to HER2 and HER3 on HER2-overexpressing breast cancer cells leads to severe impairment of PI3K signaling and reduced cell growth whereas proliferation of primary cardiomyocytes is unaffected. The enhanced coating effect of MCLA-128 also supports its ADCC activity. Citation Format: Cecile Geuijen, Eric Rovers, Tristan Gallenne, David Maussang-Detaille, Arjen Kramer, Nellie Nieuwenhuizen, Carina Clements, Katinka van Zoest, Roy Nijhuis, Therese Visser, Renate Den Blanken-Smit, Willem Bartelink, Vanessa Zondag-van der Zande, Linda Kaldenberg, Pieter-Fokko van Loo, Rob Roovers, Robert Doornbos, Leo Price, Stefan Braam, Setareh van Driel, Lex Bakker, Ton Logtenberg, John de Kruif, Mark Throsby. Mechanism of action of MCLA-128, a humanized bispecific IgG1 antibody targeting the HER2:HER3 heterodimer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-261. doi:10.1158/1538-7445.AM2015-LB-261

Abstract 2635: Targeting HER3 by interfering with its Sec61-mediated cotranslational insertion into the endoplasmic reticulum

Abstract There is increasing evidence implicating HER3 in several types of cancer, as a resilient copartner for HER2 in HER2-amplified cancers, and as a driver of resistance in many other types of cancer. However, the development of targeted therapies to inactivate HER3 function has been a challenging endeavor. Its tumor-driving functions are only sometimes driven by ligand-activation, and targeting its extracellular domain with antibody molecules has only limited effects. Its kinase domain functions in allostery, not catalysis, and we have previously shown that the classical ATP-analog class of tyrosine kinase inhibitors fail to inactivate it. Here we describe a novel approach that eliminates HER3 expression. This involves interfering with mechanisms critical for its cotranslational translocation into the endoplasmic reticulum (ER), resulting in the elimination of HER3 protein expression and loss of HER3 function. The small-molecule cotransin CT8 binds the Sec61 translocon channel in the ER membrane and prevents the signal peptide of the nascent HER3 protein from initiating its cotranslational translocation into the lumen of the ER, resulting in the degradation of HER3. Interestingly, CT8 treatment prevents HER3 translocation while the other HER proteins are unaffected. In addition, CT8 treatment suppresses the compensatory induction of HER3 that accompanies lapatinib treatment of HER2-amplified cancer cells, making for a durable suppression of HER2-HER3 signaling with the lapatinib-CT8 combination. Consistent with the effective inactivation of HER2-HER3 signaling, CT8 treatment synergistically enhances the apoptotic effects of lapatinib in HER2-overexpressing cancer cells. The target selectivities of cotransins are highly dependent on their structure and the signal sequence of targeted proteins and can be narrowed through structure-function studies. Our results thus highlight Sec61-dependent processing as a novel strategy to eliminate HER3 expression and function with considerable potential for the treatment of HER2-amplified and other types of cancers. Citation Format: Ana Ruiz-Saenz, Manbir Sandhu, Yazmin Carrasco, Rebecca L. Maglathlin, Jack Taunton, Mark M. Moasser. Targeting HER3 by interfering with its Sec61-mediated cotranslational insertion into the endoplasmic reticulum. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2635. doi:10.1158/1538-7445.AM2015-2635

Heregulin induces resistance to lapatinib-mediated growth inhibition of HER2-amplified cancer cells.

Human epidermal growth factor receptor 2 (HER2) amplification occurs in approximately 20% of gastric and gastroesophageal junction cancers in the United States and European Union. Lapatinib, a dual HER2 and epidermal growth factor receptor tyrosine kinase inhibitor, has demonstrated clinical efficacy in HER2-amplified cancer cells. However, several studies have shown that some cytokines can mediate resistance to lapatinib using their receptor tyrosine kinase (RTK) pathways. One of these, Heregulin1 (HRG1), can confer resistance to lapatinib-mediated growth inhibition in HER2-amplified breast cancer cells, but the underlying mechanisms remain unknown. Here, we investigated whether and how HRG1 causes resistance to lapatinib in gastric and gastroesophageal junction cancers in vitro. HER2-amplified gastric and gastroesophageal junction cancer cell lines were highly sensitive to lapatinib. Exposure to HRG1 together with lapatinib rescued cells from lapatinib-induced cell cycle arrest and apoptosis. Downregulation of HER3 with siRNA in the presence of HRG1 re-sensitized HER2-amplified cancer cells to lapatinib. Immunoblotting analysis indicated that HRG1 re-activated HER3 and AKT in the presence of lapatinib, which persisted for at least 72 h. Activation of HER3 and downstream AKT was mediated by residual activity of HER2. HRG1-mediated resistance could be reduced by PI3K/mTOR inhibitors or by complete inhibition of HER2. Thus, we conclude that HRG1 mediates resistance to lapatinib through HER3 and AKT activation, and that this depends on residual HER2 activity. Lapatinib in combination with anti-PI3K therapies or more potent HER2 inhibitors would improve the efficacy and avoid the emergence of resistant cells.

Abstract 546: GDC-0032 PI3K inhibitor enhances the efficacy of endocrine therapies in breast cancer cells by differentially regulating the expression of ER-targeted genes and increasing apoptosis.

Abstract Endocrine therapies such as letrozole or fulvestrant are commonly used treatment options for metastatic Hormone Receptor positive (HR+) breast cancer but patients ultimately relapse. Phosphatidylinositol 3-kinases (PI3K) regulate breast tumor cell growth, migration and survival. The alpha isoform of PI3K is frequently mutated and activated in HR+ breast cancer and has been implicated in resistance to endocrine therapies. PI3K inhibitors are therefore attractive for combination with endocrine therapies. GDC 0032 is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30 fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. Preclinical data show that GDC-0032 has increased activity against PI3K alpha isoform (PIK3CA) mutant and HER2-amplified cancer cell lines. Single agent and combination studies were carried out to determine if GDC-0032 enhances the anti-tumor activity of endocrine therapies in human breast cancer models. MCF7 cells (Estrogen Receptor positive (ER+), PI3K alpha E545K mutant) were transfected with the aromatase gene and stable clones (MCF7-ARO) were selected that are capable of converting androstenedione to estrogen in culture. When grown in the presence of androstenedione, MCF7-ARO cells were more reliant on estrogen for growth. Under these conditions the cells were treated with GDC-0032 in combination with endocrine therapies and assayed for cellular viability, modulation of PI3K pathway and ER pathway markers and apoptosis induction. The combination of GDC-0032 and endocrine therapies decreased the cellular viability of MCF7-ARO cells and increased apoptosis relative to either single agent. We observed cross-talk between the PI3K and ER pathways that suggests a mechanism of action for the combination. The data provide rationale for evaluating GDC-0032 in combination with endocrine therapies for HR+ breast cancer treatment in the clinic. Citation Format: Jeffrey J. Wallin, Jane Guan, Rebecca Hong, Carol O'Brien, Timothy Wilson, Michelle Nannini, Deepak Sampath, Marcia Belvin, Lori S. Friedman. GDC-0032 PI3K inhibitor enhances the efficacy of endocrine therapies in breast cancer cells by differentially regulating the expression of ER-targeted genes and increasing apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 546. doi:10.1158/1538-7445.AM2013-546

Abstract 3043: Akt-mTorc2 signaling underlies cellular addiction to HER3 signaling in HER2-amplified cancers.

Abstract HER2 amplified breast cancers are characterized by constitutive HER3 phosphorylation. Efforts to target the functionally relevant HER2-HER3 signaling complex in these tumor cells has proven difficult due to robust feedback signaling mechanisms that restore its signaling in the face of anything short of the complete inactivation of HER2 kinase activity. To study this in more depth, we used a combination of genetic and chemical genetic approaches designed to study the cellular adaptational responses to network perturbation. We find that positive or negative perturbation of any signaling node from upstream HER2 or HER3 down to the two mTor complexes results in compensatory responses that function to maintain homeostasis. The responses to various perturbations are mechanistically distinct, but taken together reveal an uncompromising core topology driven by a concentric Akt-mTorc2 positive-feedback loop buffered by HER3 and PHLPP nodes. While mTorc1 signaling is also subject to feedback regulation, it is not involved in the core topology and its output is not always protected in these tumors. The central role of mTor is consistent with its known functions as a master regulator of cellular homeostasis highly conserved across eukaryotes. But the inclusion of HER3 in this central homeostatic network is a tumor cell anomaly. To better delineate how this homeostatic network is altered in tumor cells, we compared and contrasted the network topology in a panel of HER2-amplified cancer cells with a panel of immortalized breast epithelial cells. Much of the feedback circuitries are preserved across tumor and untransformed cells. But the driving Akt-mTorc2 positive-feedback loop is lacking in untransformed cells. This is due to the fact that Akt drives mTorc2 signaling in tumor cells but not in untransformed cells. Consistent with a central role of mTorc2 in these tumors, knockdown of Rictor, but not Raptor, is lethal in HER2-amplified SkBr3 tumor cells. These studies reveal a key alteration of Akt-mTor signaling in HER2 driven tumors that underlies the pathological incorporation of HER3 into an mTor-driven network, thus putting the HER2-HER3 tumor driver under the protection of this evolutionarily ancient pathway, highly competent at homeostasis. Citation Format: Dhara N. Amin, Deepika Ahuja, Gregory S. Ducker, Paul Yaswen, Kevan Shokat, Mark Moasser. Akt-mTorc2 signaling underlies cellular addiction to HER3 signaling in HER2-amplified cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3043. doi:10.1158/1538-7445.AM2013-3043

Abstract LB-64: GDC-0032, a beta isoform-sparing PI3K inhibitor: Results of a first-in-human phase Ia dose escalation study.

Abstract Background: GDC-0032 is an orally bioavailable, potent, and selective inhibitor of Class I PI3Kα, δ, and γ isoforms, with 30 fold less inhibition of the PI3K β isoform relative to the PI3Kα isoform. Preclinical data show that GDC-0032 has increased activity against PI3Kα isoform (PIK3CA) mutant and HER2-amplified cancer cell lines. Methods: A Phase Ia, multicenter, open-label study was conducted to evaluate the safety and pharmacokinetics (PK) of GDC-0032 administered once daily in patients with locally advanced or metastatic solid tumors. Preliminary assessment of the anti-tumor activity of GDC-0032 was also performed. The study consisted of 3+3 dose-escalation cohorts with a 35-day window to evaluate dose-limiting toxicity (DLT). Results: As of August 1, 2012, 34 patients were enrolled in this study. Five dosing cohorts ranging from 3 to 16 mg QD were tested (3, 5, 8, 12, and 16 mg), and dose escalation has been completed. Two DLTs (Grade 4 hyperglycemia and Grade 3 fatigue) were observed in two patients from the 16 mg cohort. Adverse events (AEs) assessed by the investigator as related to GDC-0032 in ≥10% of patients, were diarrhea, hyperglycemia, fatigue, nausea, decreased appetite, and vomiting. The only Grade 4 treatment-related AE (hyperglycemia) occurred in the 16 mg cohort. GDC-0032 PK was approximately dose proportional and time independent with a mean t1/2 of 40 hours. Paired pre-treatment and on-treatment tumor biopsies of a patient in the 3 mg cohort showed pharmacodynamic inhibition of the PI3K pathway as assessed by reverse phase protein array. Metabolic partial responses via FDG-PET (≥ 20% decrease in mSUVmax) were observed in 7 out of 13 patients assessed (54%). Clinical partial responses (PRs) were observed in 5 patients treated at doses of GDC-0032 ranging from 3-12 mg QD. Consistent with GDC-0032 preclinical data, 3 PRs (2 confirmed) were observed out of 5 patients with PIK3CA mutant breast cancer (RECIST -30% to -70%). One cPR has been observed in a patient with PIK3CA mutant NSCLC, and 1 uPR in a patient with HER2-positive, PIK3CA wildtype, breast cancer. Conclusion: GDC-0032 is a PI3K inhibitor with dose-linear PK and expected in-class toxicities. Pharmacodynamic inhibition of the PI3K pathway has been observed in tumor biopsies and FDG-PET imaging. Clinical partial responses observed in patients with PIK3CA mutant and HER2+ tumors suggest that GDC-0032 may have increased anti-tumor activity in diagnostically defined subpopulations. Citation Format: Dejan Juric, Ian Krop, Ramesh K. Ramanathan, Jim Xiao, Sandra Sanabria, Timothy R. Wilson, YounJeong Choi, Hema Parmar, Jerry Hsu, Jose Baselga, Daniel D. Von Hoff. GDC-0032, a beta isoform-sparing PI3K inhibitor: Results of a first-in-human phase Ia dose escalation study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-64. doi:10.1158/1538-7445.AM2013-LB-64