HER2-amplified Breast Cancer Research Articles

The marine natural product coibamide targets expression of HER family receptors

Natural products have historically been a significant source of inspiration for drug design and development, particularly in the areas of cancer and infectious disease. The utility of natural products as drug leads is attributed to the fact that these molecules have evolved to bind specific biological targets and have the potential to reveal new aspects of cell signaling. We previously reported the discovery of coibamide, a cytotoxic N‐methyl‐stabilized depsipeptide isolated from a marine cyanobacterium growing within Coiba National Park, Panama. Initial testing of coibamide in the National Cancer Institute in vitro 60 (NCI60) cancer cell line panel revealed pM to nM potency as a growth inhibitor, an unmatched selectivity profile and histological selectivity for breast, CNS‐derived tumors, ovarian and colon cancer cells.We have undertaken studies to determine the mechanism of action of coibamide and have determined that coibamide selectively targets the ER secretory pathway. To date, these relatively rare natural products are known for their ability to selectively inhibit biosynthesis receptor tyrosine kinases, growth factors and cell adhesion proteins. We studied the relative expression of human epidermal growth factor receptor (HER) proteins in human HER2‐amplified and triple negative breast cancer cells following exposure to coibamide. Coibamide inhibited expression of HER family members with differential sensitivity (EGFR=HER3<<HER2). Time‐ and concentration‐dependent decreases in HER protein expression were accompanied by a dampening in AKT/MAPK signaling. Coibamide promoted proteasomal degradation of HER family proteins consistent with the action of an ER secretory pathway inhibitor. These results suggest that coibamide preferentially inhibits the biosynthesis of a subset of ER secretory pathway proteins to induce cancer cell death rather than a total inhibition of cellular secretory function. The HER family of proteins are associated with drug resistance and treatment failure in response to many modern, targeted anti‐cancer medicines and thus this work has the potential to reveal a new way to target HER signaling.Support or Funding InformationThis work was supported by the Oregon State University (OSU) College of Pharmacy, the American Brain Tumor Association (JEI) and an American Foundation for Pharmaceutical Education (AFPE) Pre‐Doctoral Fellowship in the Pharmaceutical Sciences (JDS)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Selective mTORC2 Inhibitor Therapeutically Blocks Breast Cancer Cell Growth and Survival.

Small-molecule inhibitors of the mTORC2 kinase (torkinibs) have shown efficacy in early clinical trials. However, the torkinibs under study also inhibit the other mTOR-containing complex mTORC1. While mTORC1/mTORC2 combined inhibition may be beneficial in cancer cells, recent reports describe compensatory cell survival upon mTORC1 inhibition due to loss of negative feedback on PI3K, increased autophagy, and increased macropinocytosis. Genetic models suggest that selective mTORC2 inhibition would be effective in breast cancers, but the lack of selective small-molecule inhibitors of mTORC2 have precluded testing of this hypothesis to date. Here we report the engineering of a nanoparticle-based RNAi therapeutic that can effectively silence the mTORC2 obligate cofactor Rictor. Nanoparticle-based Rictor ablation in HER2-amplified breast tumors was achieved following intratumoral and intravenous delivery, decreasing Akt phosphorylation and increasing tumor cell killing. Selective mTORC2 inhibition in vivo, combined with the HER2 inhibitor lapatinib, decreased the growth of HER2-amplified breast cancers to a greater extent than either agent alone, suggesting that mTORC2 promotes lapatinib resistance, but is overcome by mTORC2 inhibition. Importantly, selective mTORC2 inhibition was effective in a triple-negative breast cancer (TNBC) model, decreasing Akt phosphorylation and tumor growth, consistent with our findings that RICTOR mRNA correlates with worse outcome in patients with basal-like TNBC. Together, our results offer preclinical validation of a novel RNAi delivery platform for therapeutic gene ablation in breast cancer, and they show that mTORC2-selective targeting is feasible and efficacious in this disease setting.Significance: This study describes a nanomedicine to effectively inhibit the growth regulatory kinase mTORC2 in a preclinical model of breast cancer, targeting an important pathogenic enzyme in that setting that has been undruggable to date. Cancer Res; 78(7); 1845-58. ©2018 AACR.

338 (PB-133) - Loss of HER2 amplification and disease prognosis after neoadjuvant treatment of HER2 amplified breast cancer

338 (PB-133) - Loss of HER2 amplification and disease prognosis after neoadjuvant treatment of HER2 amplified breast cancer

Coamplification of miR-4728 protects HER2-amplified breast cancers from targeted therapy

HER2 (ERBB2) amplification is a driving oncogenic event in breast cancer. Clinical trials have consistently shown the benefit of HER2 inhibitors (HER2i) in treating patients with both local and advanced HER2+ breast cancer. Despite this benefit, their efficacy as single agents is limited, unlike the robust responses to other receptor tyrosine kinase inhibitors like EGFR inhibitors in EGFR-mutant lung cancer. Interestingly, the lack of HER2i efficacy occurs despite sufficient intracellular signaling shutdown following HER2i treatment. Exploring possible intrinsic causes for this lack of response, we uncovered remarkably depressed levels of NOXA, an endogenous inhibitor of the antiapoptotic MCL-1, in HER2-amplified breast cancer. Upon investigation of the mechanism leading to low NOXA, we identified a micro-RNA encoded in an intron of HER2, termed miR-4728, that targets the mRNA of the Estrogen Receptor α (ESR1). Reduced ESR1 expression in turn prevents ERα-mediated transcription of NOXA, mitigating apoptosis following treatment with the HER2i lapatinib. Importantly, resistance can be overcome with pharmacological inhibition of MCL-1. More generally, while many cancers like EGFR-mutant lung cancer are driven by activated kinases that when drugged lead to robust monotherapeutic responses, we demonstrate that the efficacy of targeted therapies directed against oncogenes active through focal amplification may be mitigated by coamplified genes.

Abstract P6-13-07: Chemotherapy with and without trastuzumab or no treatment in elderly patients with HER2 amplified breast cancer at a single center

Abstract Introduction Trastuzumab with systemic chemotherapy has shown an improvement in outcomes for patients (pts) with HER2 amplified/overexpressed (HER2+) breast cancer. Pts enrolled onto trials were young with a minority of pts at ≥65 years (yrs) of age. Herein, we report the administration of systemic treatment (ST) (chemotherapy and/or trastuzumab) verus no treatment in elderly pts at a single center. Methods Patients ≥65 yrs with stage I-III HER2+ (defined as IHC 3+ or FISH >2.0) breast cancer, treated at Memorial Sloan Kettering Cancer Center between 2000-2012, were retrospectively identified from our database. Clinicopathologic features were retrieved and co-morbidity indexes (CI) were calculated. Pts were divided by hormone receptor (HR) (defined as ER >10% and/or PR >10%) status into HER2+HR- and HER2+HR+. Each group was further divided by use of ST into: chemotherapy and trastuzumab (CT+T), chemotherapy alone (CT) or no systemic treatment (No Rx). Patients receiving neoadjuvant ST or trastuzumab only as ST were excluded from the KM analysis. Primary objective was to identify patterns of treatment recommendation in the elderly population. We explored disease-free survival (DFS) as estimated using the Kaplan-Meier (KM) method. Results We identified 300 pts ≥65 yrs with HER2+ tumors. 128 (42.7%) were HER2+HR- and 172 (57.3%) were HER2+HR+. The median follow-up for all patients was 6.1 years (range, 0.07-16.7). In the HER2+HR- group, 63 (49.2%) patients received CT+T, 25 (19.5%) CT alone, and 40 (31.3%) had no Rx. Anthracycline based chemotherapy was administered to 57/88 (65%) of patients on CT. Women receiving chemotherapy with or without trastuzumab were younger (65-70 vs >70 years of age) (p=.002) and had more advanced tumor stages (p=.003). Their respective 5-yr DFS KM estimates were 0.84, 0.80, and 0.61 (logrank p=0.06). In the HER2+HR+ group, 77 (44.8%) patients received CT+T, 22 (12.8%) CT alone, and 73 (42.2%) had no Rx. Anthracycline based chemotherapy was administered to 51/99 (51%) of patients on CT. Endocrine therapy was given to 153/172 (89%) of the total cohort. Women receiving chemotherapy with or without trastuzumab were younger (p<.001), and had higher nuclear grade (NG) (p=.04), more lymphovascular invasion (<.001) and more advanced tumor stages (p=.002). Their respective 5-yr DFS KM estimates were 0.84, 1.00, and 0.83 (log rank p=0.02). Conclusions At a single center, in the elderly populations at ≥65 years of age with HER2+ HR- and HER2+HR+ breast cancer, pts who received systemic treatment were younger and had higher stage of disease than those who received no treatment. In an exploratory analysis, there appeared to be a benefit of systemic treatment in pts in the HER+HR- group. Citation Format: Muhsen S, Dang C, Plitas G, Seier K, Stempel M, Patil S, Morrow M, El-Tamer M. Chemotherapy with and without trastuzumab or no treatment in elderly patients with HER2 amplified breast cancer at a single center [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-13-07.

Abstract PD3-03: Circulating tumor DNA in HER2 amplified breast cancer: A translational research substudy of the NeoALTTO phase 3 trial

Abstract Purpose. To evaluate whether circulating tumor DNA (ctDNA) was associated with response to neoadjuvant anti-HER2 targeted therapies (NAT) in patients enrolled in the Neoadjuvant Lapatinib and/or Trastuzumab Treatment Optimization (NeoALTTO) trial (BIG 1-06). Methods. Cell-free DNA from plasma collected before NAT, at week 2 and before surgery were processed using patient-specific droplet digital polymerase chain reaction (ddPCR) assays for the detection of PIK3CA and p53 mutations. These mutations had been previously detected in baseline primary tumor biopsy samples using mass spectrometry-based genotyping, exome or RNA sequencing. Chi-square test was used to evaluate differences in ctDNA detection according to the NeoALTTO stratification factors: clinical tumor size (T2 vs T3/4), clinical nodal status (N0/1 vs N2), Hormone Receptor status, HR (positive vs negative) and type of breast surgery planned (conservative vs mastectomy). Associations between ctDNA detection and either pathological complete response (pCR) or event-free survival (EFS) after adjustment for the NeoALTTO stratification factors were investigated using logistic and Cox regression models, respectively. Results. A total of 69 of 455 (15.2%) patients enrolled in the NeoALTTO trial had either PIK3CA (55 patients) or p53 (14 patients) mutations detected in the baseline tumor samples and at least one plasma sample with evaluable ctDNA results. Out of the 69 patients, 36 (52.2%) had HR-negative disease, 45 (65.2%) had T2 tumors, 55 (79.7%) N0/1 disease, 50 (72.5%) were candidates for mastectomy and 27 (39.1%) received trastuzumab and lapatinib in combination. ctDNA before NAT, at week 2 and before surgery was detected in 29 (42%) of 69, in 13 of 64 patients (20.3%) and in 4 of 59 (6.7%) patients analyzed, respectively. There was no significant difference in ctDNA detection before NAT according to HR status, clinical tumor size, nodal status or type of planned breast surgery. After adjustment for the NeoALTTO stratification factors, ctDNA detection before NAT was associated with decreased odds of achieving pCR, (odds ratio, OR 0.16, 95% CI 0.04-0.71, p=0.016), but not with EFS (Hazard Ratio 1.32, 95%CI 0.74-2.37, p=0.345). Neither ctDNA detection at week 2 nor before surgery was significantly associated with either pCR or EFS but these analyses were underpowered. Conclusion In patients with HER2-amplified tumors and either PIK3CA or p53 mutations, detection of ctDNA before neoadjuvant anti-HER2 targeted therapies is associated with decreased probability of pCR. Citation Format: Ignatiadis M, Silva MJ, Campbell C, Bradbury I, de Azambuja E, Maetens M, Fumagalli D, Rodrik-Outmezguine V, Di Cosimo S, Rosa D, Chia S, Wardley A, Ueno T, Janni W, Huober J, Baselga J, Piccart M, Sotiriou C, Loi S, Rothé F, Dawson S. Circulating tumor DNA in HER2 amplified breast cancer: A translational research substudy of the NeoALTTO phase 3 trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-03.

Abstract P5-10-01: Cellular senescence within HER2-amplified breast cancer: Potential implications for breast cancer immune surveillance and HER2-targeted therapy resistance

Abstract Background:Oncogene-induced senescence is considered as a barrier to tumor progression that arrests cells at risk for malignant transformation. Nevertheless, numerous findings demonstrate that senescent cells may also have the opposite function and promote tumor progression through the release of multiple factors called the senescence-associated secretory phenotype or senescence secretome. It is likely that the composition and the physiological consequences mediated by the senescence secretome are dependent on the oncogenes that trigger the senescence program. Breast cancer represents a heterogenous disease that can be divided into breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities. Therefore, tumor initiation and progression of breast cancer subtypes is triggered by variable oncogenic stimuli, and differences in the senescence secretomes within breast tumors might be responsible for tumor initiation, progression, metastasis and therapeutic response. Beside many studies concerning the role of senescence as a barrier to tumor progression using murine xenograft models very few investigations have been performed to elucidate how often senescent tumor cells appear within untreated human tumors, and if present whether these senescent tumor cells may play a role in disease progression, cancer immunosurveillance and therapy resistance. Methode: In the present study we analysed the appearance of senescent cells within invasive human breast cancers from 129 untreated patients. Cellular senescence was detected by the use of SAβ-gal staining and by immunohistochemical detection of p16, p21, p53, Ki67 and lamin B1. Results: Detection of cellular senescence by the use of SAβ-gal staining and detection of p16, p53, Ki67 and lamin B1 within invasive breast carcinomas indicate that senescent tumor cells varies strongly according to the breast cancer subtype. The highest percentages of senescent tumor cells exist within in the HER2-positive and luminal A breast carcinomas whereas no or very few senescent tumor cells were found in triple negative breast tumors. Based on these findings we suggest that the composition of secretomes released by senescent tumor cells from different breast cancer subtypes might be very distinct in respect to their ability to recruit immune cells, which can eliminate senescent tumor cells on one hand and regulate tumor growth, immune surveillance and therapy resistance on the other. Conclusion: Further characterization of senescent secretomes from HER2 and other breast cancer subtypes and their potential role in tumor progression, immune surveillance and therapy response might be warranted for the understanding of cancer biology as well as prognostic and therapeutic applications. Citation Format: Thaler S, Schad A, Kirkpatrick CJ, Sleeman JP, Springer E, Schmidt M, Cotarelo CL. Cellular senescence within HER2-amplified breast cancer: Potential implications for breast cancer immune surveillance and HER2-targeted therapy resistance [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-10-01.

Abstract P3-07-07: The pan-HER inhibitor, neratinib and wingless-type MMTVs (Wnt)/Wnt regulators in human breast cancer; a biological and clinical perspective

Abstract Background. Neratinib is an orally available tyrosine kinase inhibitor that irreversibly binds and inhibits EGFR, HER2 and HER4 receptor tyrosine kinases. Neratinib has been shown to have clinical activity in HER2-amplified or overexpressed breast cancers and those with HER2 mutations. However, there are indications that it may also work on other subtypes that are not strongly positive for the receptors. The present study first screened the effects of neratinib on a range of kinase targets and identified that the Wnt signalling components are key factors that allow neratinib to interact and targets. These targets were also validated in a cohort of human breast cancer. Methods. Human breast cancer cohorts (n=124) were tested for the transcript expression of HER family including EGFR, HER2, HER3 and HER4 and a number of Wnt family members and the Wnt signalling regulators, ie. GSK3, Axin-1, Axin-2 and β-catenin. The expression patterns were analysed against the clinicopathological and survival status of the patients. Neratinib was tested on a panel of breast cancer cell lines including triple negative cells for the effects on cytoxicity, cell growth, matrix adhesiveness and cellular migration. Signalling kinase pathways were screened using an antibody based kinase array. The effect of neratinib on multiple protein kinases was tested on the cell models, together with other kinase inhibitors. Results. Neratinib had an inhibitory effect on the cellular migration and cell-matrix adhesiveness of breast cancers at non-toxic concentrations, an effect more profound with MCF-7 and T47D cell lines than with BT20 and MDA MB-231 which are negative for the ER/EGFR/HER2 receptors. Of the multiple kinase inhibitors tested, neratinib was found to exert inhibition on cell function in synergy with the Wnt/β-catenin inhibitor (FK535) and GSK3 inhibitor (TWS119). The expression of the HER and Wnt family members, Wnt Inhibitory Factor-1 and Wnt regulators varied in mammary and breast cancer tissues and in their correlation with the clinicopathological factors. Of the aberrantly expressed receptors, Wnts and Wnt regulators, HER-2 and 4 were found to significantly correlate with Wnt10b (p<0.05), EGFR/HER1 was found significantly correlated with GSK3 (p<0.05) and HER-3 with Wnt5a (p<0.03). Furthermore, we identified that the integrated expression pattern of five of these factors, namely EGFR, HER2, HER4, Wnt5 and Wnt Inhibitory Factor-1 formed an expression signature that were significantly linked to the overall survival (survival time 148±3.7 vs 113.7 ±7.5 months for favourable and non-favourable pattern, respectively, p=0.002) and disease free survival (p=0.004) of the patients (median follow-up 120 months) Conclusion. Neratinib, at non-toxic concentration levels, is a profound inhibitor of the migration and matrix adhesion of breast cancer cells, cell functions linked to the aggressiveness and metastasis of breast cancer cells. Its synergistic effects with the Wnt and GSK3 inhibitor, together with the prognostic value of the HER family/Wnt, indicate that the Wnt pathway together with the HER family forms a new molecular indicator and target when considering neratinib in the treatment patients with breast cancer. Citation Format: Owen S, Ruge F, Lalani AS, Avogadri-Connors F, Bryce RP, Davies E, Jiang WG. The pan-HER inhibitor, neratinib and wingless-type MMTVs (Wnt)/Wnt regulators in human breast cancer; a biological and clinical perspective [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-07.

Abstract P2-09-25: Clinical and pathologic characteristics of breast cancers determined to be HER2-positive by fluorescence in-situ hybridization (FISH) using alternative chromosome 17 probes

Abstract Background: Based on updated 2013 ASCO/CAP guideline for HER2 testing, cases with a HER2/CEP17 ratio < 2.0 but with an average HER2 copy number > 4.0 and <6.0 signals/cell are considered equivocal. In such cases, HER2 testing using alternative chromosome 17 probes was proposed as one way to resolve the equivocal FISH results. Using the alternative probe method increases the number of cancers categorized as HER2 positive but brings to question if these cancers truly represent HER2 amplified breast cancers and derive the same benefit from anti-HER2 therapies. Methods: Since 2013, all breast cancers at our institution that were HER2 equivocal by traditional FISH but classified as HER2 positive using the alternative probe method were assessed for clinical and pathologic features including histologic type and grade, TNM stage, HER2: alternative probe ratio, treatment, and clinical outcome. Results: We identified 24 invasive breast cancers considered HER2 positive by the alternative probe method: 23 (96%) were estrogen receptor-positive (ER+) and 20 (83%) were progesterone receptor- positive. Histologically, only 2 were invasive lobular carcinomas; all others were ductal or had ductal and lobular features. Most cancers (63%) had low or intermediate histologic grade: Grade 1 (n=3); Grade 2 (n=12); Grade 3 (n=9). Clinical information was available for 18 patients: 2 had metastatic disease, 1 had a local recurrence after mastectomy and 15 patients had early stage disease; 9 with node negative disease and 6 with nodal involvement. HER2 IHC was equivocal (2+) in 16 (66.7%) cases, positive (3+) in 4 (16.7%) cases, and negative (0 or 1+) in 4 (16.7%) cases. The average HER2 copy number was 4.77, the average HER22:p53 ratio was 2.61. Repeat HER2 testing on a 2nd tumor sample was performed in 8 cases: HER2-positivity was confirmed in only 2 (25%) cases and by the alternative probe only. Treatment information was available for 17 patients: 1 had T1aN0M0 lesion and did not get chemotherapy, 16 received chemotherapy and 13 received trastuzumab-based chemotherapy. Eleven patients with early stage disease received chemotherapy and trastuzumab. Of these patients, 10/11 were ER+, 7/11 were node negative and 5/11 had grade 2 tumors, yet only one tumor was assessed by oncotype recurrence score ( RS = 29). Three patients received chemotherapy and trastuzumab in the neoadjuvant setting: 1 had a complete pathologic response, 1 a partial response, and 1 has not yet gone to surgery. One additional patient received neoadjuvant chemo alone and achieved a partial response. Conclusions: Breast cancers considered HER2+ by the alternative probe method but not by traditional FISH are almost always ER-positive and most have low or intermediate histologic grade. Repeat HER2 testing on a subsequent tumor sample did not confirm HER2-positivity in 75% of cases. Almost all patients with early stage disease received chemotherapy and trastuzumab based on the alternative probe results without molecular assessment to predict chemotherapy response. Intrinsic molecular subtyping using PAM50 analysis on these cancers is underway to determine how many are HER2-enriched by molecular assessment. Citation Format: Desai NV, Torous V, Cruz C, Schnitt SJ, Tung N. Clinical and pathologic characteristics of breast cancers determined to be HER2-positive by fluorescence in-situ hybridization (FISH) using alternative chromosome 17 probes [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-25.

Potential Management of Circulating Tumor DNA as a Biomarker in Triple-Negative Breast Cancer.

As a specific subtype of breast cancer, Triple-negative breast cancer (TNBC) is associated with worse prognosis and higher tumor aggressiveness than HER2-amplified or hormone receptor positive breast cancers. Circulating tumor DNA (ctDNA), as a non-invasive “liquid biopsy”, is an emerging original blood-based biomarker for early breast cancer diagnosis, monitoring treatment response, and determining prognosis. In TNBC patients, ctDNA has an inherent tendency to characterize tumor heterogeneity and metastasis-specific mutations providing a key alternative to tumor tissue profiling. Several studies have already demonstrated the potential of ctDNA in TNBC patients from early to advanced stages of the disease including diagnosis, therapy decisions and assessment of prognosis. This review provides a critical brief summary of the evidence that gives credence to the utility of ctDNA as a biomarker for its role into clinical management in TNBC.

Abstract B169: Neratinib has clinical activity in HER2-amplified breast cancer patients with tumors that have acquired activating mutations in HER2

Abstract Overexpression/amplification of HER2/ERBB2 occurs in 20% of breast cancers. Thanks to specific anti-HER2 agents, the prognosis of HER2-positive breast cancer has improved considerably. However, acquired resistance inevitably emerges over time and tumors escape pharmacologic pressure. In this work, we propose that acquisition of activating somatic mutations in HER2 upon anti-HER2 therapy may be more frequent than commonly reported and can reduce sensitivity to these agents. Moreover, we tested whether neratinib, an irreversible pan-HER inhibitor, is effective in tumors bearing both amplification and mutations of ERBB2. By targeted exome sequencing, we found that samples from metastatic breast cancer (MBC) patients relapsing to multiple lines of anti-HER2 therapy presented the acquisition of HER2 mutations. These mutations spanned from the extracellular domain (L313I, R456C) to the kinase domain (L755S, D769Y) of the receptor. To investigate the role of these mutations in drug resistance, we conducted functional studies by stably transducing the L755S HER2 mutation (the most frequent HER2 mutation in breast cancer) in two ERBB2-amplified breast cancer cell lines intrinsically sensitive to HER2 inhibition. In both models, we found that expression of L755S mutant-HER2 was sufficient to limit sensitivity to trastuzumab, lapatinib, or the combination of both agents. Consistently, neither trastuzumab nor lapatinib was effective in inhibiting tumor growth of patient-derived xenografts established from a patient with ERBB2-amplified/mutant (D769Y) breast cancer. However, neratinib treatment demonstrated marked sensitivity in this tumor model, resulting in significant tumor growth inhibition. The antitumor activity of neratinib was also explored in breast cancer patients with coexisting ERBB2 amplification and mutation, either by compassionate use after failure of standard-of-care therapy or as part of a “basket” trial (NCT01953926) enrolling ERBB2-mutant patients. In both settings, we observed durable clinical response to neratinib. MBC case #HER2 co-mutationResponse to neratinibDurability of response (mo)1D769YSD62L313ISD93Y772_A775dupSD44L755SSD55V777LPR6 Our findings indicate that acquired HER2 mutations may reduce the effectiveness of therapeutic agents commonly used for the management of ERBB2-amplified MBC. Moreover, we propose neratinib as an effective treatment option for patients whose tumors harbor both ERBB2 amplifications and mutations. Citation Format: Emiliano Cocco, F. Javier Carmona, Helen H. Won, Michael F. Berger, David M. Hyman, Valentina Rossi, Carmen Chan, Alyssa Moriarty, Kyriakos P. Papadopoulos, Michael J. Wick, James Cownie, Ivana Sarotto, Richard E. Cutler, Francesca Avogadri-Connors, Peter Savas, Alshad S. Lalani, Valentina Boni, Sherene Loi, Jose Baselga, Filippo Montemurro, Maurizio Scaltriti. Neratinib has clinical activity in HER2-amplified breast cancer patients with tumors that have acquired activating mutations in HER2 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B169.

Structure-Activity Relationships of New Natural Product-Based Diaryloxazoles with Selective Activity against Androgen Receptor-Positive Breast Cancer Cells.

Targeted therapies for ER+/PR+ and HER2-amplified breast cancers have improved patient survival, but there are no therapies for triple negative breast cancers (TNBC) that lack expression of estrogen and progesterone receptors (ER/PR), or amplification or overexpression of HER2. Gene expression profiling of TNBC has identified molecular subtypes and representative cell lines. An extract of the Texas native plant Amyris texana was found to have selective activity against MDA-MB-453 cells, a model of the luminal androgen receptor (LAR) subtype of TNBC. Bioassay-guided fractionation identified two oxazole natural products with selective activity against this cell line. Conducted analog synthesis and structure-activity relationship studies provided analogs with more potent and selective activity against two LAR subtype cell line models, culminating in the discovery of compound 30 (CIDD-0067106). Lead compounds discovered have potent and selective antiproliferative activities, and mechanisms of action studies show they inhibit the activity of the mTORC1 pathway.

Adaptation to TKI Treatment Reactivates ERK Signaling in Tyrosine Kinase-Driven Leukemias and Other Malignancies.

FMS-like tyrosine kinase-3 (FLT3) tyrosine kinase inhibitors (TKI) have been tested extensively to limited benefit in acute myeloid leukemia (AML). We hypothesized that FLT3/internal tandem duplication (ITD) leukemia cells exhibit mechanisms of intrinsic signaling adaptation to TKI treatment that are associated with an incomplete response. Here, we identified reactivation of ERK signaling within hours following treatment of FLT3/ITD AML cells with selective inhibitors of FLT3. When these cells were treated with inhibitors of both FLT3 and MEK in combination, ERK reactivation was abrogated and anti-leukemia effects were more pronounced compared with either drug alone. ERK reactivation was also observed following inhibition of other tyrosine kinase-driven cancer cells, including EGFR-mutant lung cancer, HER2-amplified breast cancer, and BCR-ABL leukemia. These studies reveal an adaptive feedback mechanism in tyrosine kinase-driven cancers associated with reactivation of ERK signaling in response to targeted inhibition. Cancer Res; 77(20); 5554-63. ©2017 AACR.

HER2 Reactivation through Acquisition of the HER2 L755S Mutation as a Mechanism of Acquired Resistance to HER2-targeted Therapy in HER2+ Breast Cancer.

Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies.Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition.Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2-irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivoConclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123-34. ©2017 AACR.

Synergistic action of the MCL-1 inhibitor S63845 with current therapies in preclinical models of triple-negative and HER2-amplified breast cancer.

The development of BH3 mimetics, which antagonize prosurvival proteins of the BCL-2 family, represents a potential breakthrough in cancer therapy. Targeting the prosurvival member MCL-1 has been an area of intense interest because it is frequently deregulated in cancer. In breast cancer, MCL-1 is often amplified, and high expression predicts poor patient outcome. We tested the MCL-1 inhibitor S63845 in breast cancer cell lines and patient-derived xenografts with high expression of MCL-1. S63845 displayed synergistic activity with docetaxel in triple-negative breast cancer and with trastuzumab or lapatinib in HER2-amplified breast cancer. Using S63845-resistant cells combined with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9) technology, we identified deletion of BAK and up-regulation of prosurvival proteins as potential mechanisms that confer resistance to S63845 in breast cancer. Collectively, our findings provide a strong rationale for the clinical evaluation of MCL-1 inhibitors in breast cancer.

Housekeeping gene as a source of calibrator candidate for HER-2 scoring in frozen tissue breast cancer study based on qPCR

Rismaya, Azamris, Budiarti S, Desriani. 2017. Housekeeping gene as a source of calibrator candidate for HER-2 scoring in frozen tissue breast cancer study based on qPCR. Biodiversitas 18: 1041-1046. HER-2 amplification in breast cancer gives an implication in therapy and prognosis. FDA have been approved Fluorescence In situ hybridization (FISH) for HER-2 amplification and immunohistochemistry (IHC) for protein expression measurement. In this recently times, quantitative PCR techniques have been reported as an alternative method for HER-2 amplification determination. Here in this report, we investigated new reference gene coming from housekeeping gene as an alternative calibrator for HER-2 amplification scoring based on qPCR methods. We compared two recommended housekeeping gene, 18S rRNA and β-Actin, as a candidate for a HER-2 calibrator. For methods development, optimization and comparison study between 18S rRNA and β-Actin, we used non-invasive samples (DNA buccal cells). Selected candidate reference further was tested to eighteen breast cancer sample, validated with qPCR methods refer to Mendoza et al. 2013. β- Actin is shown as the best candidate for HER-2 scoring status determination. qPCR concordance result with other qPCR methods refers to Mendoza et al. 2013 shown 94.4%. The qPCR efficiency and % CV value shown as a requirement as a theory. qPCR efficiency was 103.8-105.3% for β-Actin and HER-2 respectively while CV value was around 1.2%. Our result showed a significant correlation with reported methods, which could potentially complement with FDA approved methods.

Abstract 3082: Deficient NOXA in HER2-amplified breast cancer drives kinase inhibitor resistance

Abstract The purpose of this study is the development of a novel combination therapy that targets HER2-amplified breast cancer. About one quarter of breast cancers harbor amplification of HER2. HER2 is a transmembrane receptor tyrosine kinase (RTK) belonging to the ERBB family of receptors (ERBB1-4). Upon hetero- and homo-dimerization, HER2 activates several key intracellular pathways, regulating many cellular functions including proliferation and survival. HER2 inhibitors (HER2i) (e.g. the receptor tyrosine kinase (RTK) inhibitor, lapatinib) are now part of standard care for treating HER2-amplified breast cancers. However, despite their anti-cancer benefit, these drugs have limited efficacy as monotherapies, which contrasts to other RTK inhibitors in other RTK-driven cancers. To better understand this apparent dichotomy, in the present study, we evaluated potential modifiers of HER2i therapy. Here, we found that the pro-apoptotic NOXA, a member of the B-cell CLL/lymphoma 2 (BCL2) family which acts mainly via inhibitory binding of the pro-survival MCL-1, was markedly down-regulated in breast cancers compared to other cancers, and this was largely attributed to the HER2-amplified subset. Experimentally, overexpressing NOXA or silencing MCL-1 dramatically sensitizes HER2 amplified breast cancer cell lines to lapatinib via apoptosis. Consistently, pharmaceutical inhibition of MCL-1 sensitizes HER2-amplified breast cancers to lapatinib in vitro and in vivo. Mechanistically, disruption of MCL-1:BIM complexes and MCL-1:BAK underlie dual HER2i/MCL-1i therapy. Therefore, deficient NOXA expression constitutes a bonafide apoptotic block in HER2 amplified breast cancers, contributes to mitigated HER2i responses, and presents a rational combination therapy that may improve HER2i responses. Citation Format: Konstantinos V. Floros, Kyung-A Song, Timothy L. Lochmann, Mark T. Hughes, Daniel A. Heisey, Hisashi Harada, Bin Hu, Jennifer Koblinski, Andrew J. Souers, Joel D. Leverson, Anthony C. Faber. Deficient NOXA in HER2-amplified breast cancer drives kinase inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3082. doi:10.1158/1538-7445.AM2017-3082

Abstract 5008: The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation

Abstract Brain metastases represent a devastating progression of luminal breast cancer. While targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models that faithfully recapitulate the disparate clinical responses in these microenvironments, we observed that brain metastases evade PI3K inhibition despite efficient drug delivery. In comparison to extracranial disease, there is increased HER3 expression and phosphorylation in the brain lesions. HER3 blockade overcomes the resistance of both HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, leading to striking tumor growth delay and significant improvement of mouse survival. Collectively, these data provide a mechanistic basis underlying therapeutic resistance in the brain microenvironment and identify rapidly translatable treatment strategiesfor HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases. Citation Format: Gino B. Ferraro, David P. Kodack, Vasileios Askoxylakis, Qing Sheng, Mark Badeaux, Shom Goel, Xiaolong Qi, Ram Shankaraiah, Alexander Z. Cao, Rakesh R. Ramjiawan, Divya Bezwada, Bhushankumar Patel, Youngchul Song, Carlotta Costa, Kamila Naxerova, Christina Wong, Jonas Kloepper, Rita Das, Angela Tam, Jantima Tanboon, Dan G. Duda, Ryan C. Miller, Marni B. Siegel, Carey K. Anders, Melinda Sanders, Valeria M. Estrada, Robert Schlegel, Carlos L. Arteaga, Elena Brachtel, Alan Huang, Dai Fukumura, Jeffrey A. Engelman, Rakesh K. Jain. The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5008. doi:10.1158/1538-7445.AM2017-5008

Abstract 1842: Breast cancer brain metastasis (BCBM) model for determination of therapeutic brain penetration

Abstract Purpose Brain metastases are presenting an increasing problem in the clinic, and especially in treatment of patients with human epidermal growth factor receptor-2 (HER2)-amplified breast cancer. Although extracranial metastases respond well to HER2 inhibitors, human clinical data shows brain metastases hide behind an intact blood-brain barrier (BBB) and are largely untreatable. Many current preclinical models lack this barrier integrity, limiting their utility in understanding delivery of drugs to the brain. We present here the development of a new model suitable for evaluating therapeutic brain penetration in addition to efficacy. Experimental procedures Human HER2-amplified BT-474 breast cancer cells were inoculated intravenously (tail vein) in female Rag2-/-;Il2rg-/- mice (2 million cells/mouse) to induce multiorgan metastasis. Formation of metastatic brain lesions was monitored by magnetic resonance imaging (MRI). For comparison, BT-474 cells were inoculated intracranially for direct brain tumor implantation (50,000 cells/mouse). Response to a suite of HER2-targeted therapies known not to appreciably cross an intact BBB (trastuzumab, lapatinib, etc.) was monitored by MRI for both metastatic and implanted brain tumors. Mice were sacrificed following signs of prolonged distress or loss of >20% body weight. Organs were collected for standard histological and immunohistochemical analysis, as well as for CLARITY tissue clearing and large-scale 3D macromolecule mapping. Results Intravenous inoculation of BT-474 cells into Rag2-/-;Il2rg-/- mice consistently reproduced the full metastatic profile seen in humans, with metastases in the lung, bone, liver, ovary, lymph, and brain tissues. Brain metastases were detected in >90% of mice inoculated intravenously. Histological analysis of metastatic brain tumors showed different morphologies and invasive characteristics compared to those intracranially implanted. Additional differences in vasculature between metastatic and implanted brain tumors were identified by CLARITY. Importantly, HER2-targeted therapy markedly delayed progression of implanted brain tumors, but failed to control metastatic brain tumor growth, recapitulating the clinical situation. Conclusions These data, together with ongoing efforts to further characterize therapeutic transport to these brain tumors, suggest that intracranial inoculation disrupts the BBB and creates artificial routes for therapeutics to reach implanted brain lesions, resulting in anomalous tumor response. In contrast, this new metastatic model reproduces the discordant effects of HER2-targeted therapy in patients, and offers a platform for studying the efficiency of therapeutic delivery across an intact BBB as well as antitumor activity, both of which are critical to effective clinical translation. Citation Format: Emily A. Wyatt, Mark E. Davis. Breast cancer brain metastasis (BCBM) model for determination of therapeutic brain penetration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1842. doi:10.1158/1538-7445.AM2017-1842

Abstract 33: The binding mode of the bispecific anti-HER2xHER3 antibody MCLA-128 is responsible for its potent inhibition of HRG-driven tumorigenesis

Abstract Introduction: MCLA-128 is as an ADCC-enhanced IgG1 bispecific antibody that targets the HER2:HER3 dimer and is currently being tested in Phase I/II clinical trials. MCLA-128 demonstrates an in vitro potency superior to other anti-HER2 and anti-HER3 antibodies in cells stimulated with high concentrations of heregulin (HRG) thereby overcoming one of the resistance mechanisms of current HER2 therapies. This study investigates the binding mode of MCLA-128 and proof of concept studies in HRG-driven tumor models. Methods: Alanine scanning shotgun mutagenesis was used to map the epitopes of MCLA-128 to HER2 and HER3. Fab fragments of MCLA-128 were crystallized with the soluble extracellular domains of HER2 and HER3. SAXS analysis on the HER2-HER3-MCLA-128 complex was performed to investigate the binding mode of the bispecific antibody in solution. Ligand-induced dimer specificity was investigated with PathHunter® heterodimerization assays. Bispecific anti-HER2xHER3 antibody and its parental anti-HER3 monoclonal antibody were labelled with 64Cu to compare their biodistribution profiles. The efficacy of MCLA-128 in HRG-driven systems was shown in vitro in MDA-MB-175 cells and in vivo in an orthotopic intracranial patient-derived xenograft (PDX) model originating from a breast cancer brain metastasis Results: The shotgun mutagenesis study identified that the bispecific antibody MCLA-128 binds amino acids T144, R166, R181 in HER2 domain I and R426 in HER3 domain III. Crystallographic studies confirmed the involvement of these critical residues and suggested that MCLA-128 locks the HER3 receptor in its ligand-unbound inactive confirmation. SAXS analysis suggests that the bispecific antibody MCLA-128 forms inter-dimer rather than intra-dimer interactions. In vitro, MCLA-128 specifically blocked HRG-induced signaling of HER2:HER3 but not HER2:HER4 heterodimers. Biodistribution of MCLA-128 in a xenograft model of breast cancer showed that the penetration of MCLA-128 in JIMT-1 HER2-amplified tumors is HER2-dependent despite the high affinity of the HER3 Fab arm for its receptor. MCLA-128 efficiently blocked tumor growth of the HRG-driven HER2 (1+) breast cancer cell line MDA-MB-175 in 3D in vitro. Treatment of orthotopically transplanted HER2-amplified breast cancer brain tumors in mice led to 100% survival with MCLA-128, in contrast to 38% and 0% survival in T-DM1 and vehicle treated mice respectively. Conclusion: MCLA-128 targets HER2-positive tumors via its HER2 arm and locks HER3 in an inactive confirmation. The potent anti-proliferative activity of MCLA-128 in vitro and in vivo supports the clinical development of this bispecific HER2xHER3 antibody in HRG-driven tumors. Citation Format: David Maussang-Detaille, Camilla de Nardis, Linda Hendriks, Carina Bartelink-Clements, Eric Rovers, Tristan Gallenne, Robert Doornbos, Lex Bakker, John de Kruif, Ton Logtenberg, Piet Gros, Cecile Geuijen, Mark Throsby. The binding mode of the bispecific anti-HER2xHER3 antibody MCLA-128 is responsible for its potent inhibition of HRG-driven tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 33. doi:10.1158/1538-7445.AM2017-33