HER2 Protein Levels Research Articles

CMTM6 overexpression confers trastuzumab resistance in HER2-positive breast cancer

Human epidermal growth factor receptor 2-positive (HER2+) breast cancer is characterized by invasive growth, rapid metastasis and chemoresistance. Trastuzumab is an effective treatment for HER2+ breast cancer; however, trastuzumab resistance leads to cancer relapse and metastasis. CKLF-like MARVEL transmembrane domain-containing 6 (CMTM6) has been considered as a new immune checkpoint for tumor-induced immunosuppression. The role of CMTM6 in trastuzumab resistance remains unknown. Here, we uncover a role of CMTM6 in trastuzumab-resistant HER2+ breast cancer. CMTM6 expression was upregulated in trastuzumab-resistant HER2+ breast cancer cell. Patients with high CMTM6 expressing HER2+ breast cancer had worse overall and progression-free survival than those with low CMTM6 expression. In vitro, CMTM6 knockdown inhibited the proliferation and migration of HER2+ breast cancer cells, and promoted their apoptosis, while CMTM6 overexpression reversed these effects. CMTM6 and HER2 proteins were co-localized on the surface of breast cancer cells, and CMTM6 silencing reduced HER2 protein levels in breast cancer cells. Co-immunoprecipitation revealed that CMTM6 directly interacted with HER2 in HER2+ breast cancer cells, and CMTM6 overexpression inhibited HER2 ubiquitination. Collectively, these findings highlight that CMTM6 stabilizes HER2 protein, contributing to trastuzumab resistance and implicate CMTM6 as a potential prognostic marker and therapeutic target for overcoming trastuzumab resistance in HER2+ breast cancer.

HER2 mRNA Levels, Estrogen Receptor Activity and Susceptibility to Trastuzumab in Primary Breast Cancer.

While the results thus far demonstrate the clinical benefit of trastuzumab in breast cancer (BC), some patients do not respond to this drug. HER2 mRNA, alone or combined with other genes/biomarkers, has been proven to be a powerful predictive marker in several studies. Here, we provide evidence of the association between HER2 mRNA levels and the response to anti-HER2 treatment in HER2-positive BC patients treated with adjuvant trastuzumab and show that this association is independent of estrogen receptor (ER) tumor positivity. While HER2 mRNA expression was significantly correlated with HER2 protein levels in ER-negative tumors, no correlation was found in ER-positive tumors, and HER2 protein expression was not associated with relapse risk. Correlation analyses in the ER-positive subset identified ER activity as the pathway inversely associated with HER2 mRNA. Associations between HER2 levels and oncogene addiction, as well as between HER2 activation and trastuzumab sensitivity, were also observed in vitro in HER2-positive BC cell lines. In ER-positive but not ER-negative BC cells, HER2 transcription was increased by reducing ligand-dependent ER activity or inducing ER degradation. Accordingly, HER2 mRNA levels in patients were found to be inversely correlated with blood levels of estradiol, the natural ligand of ER that induces ER activation. Moreover, low estradiol levels were associated with a lower risk of relapse in HER2-positive BC patients treated with adjuvant trastuzumab. Overall, we found that HER2 mRNA levels, but not protein levels, indicate the HER2 dependency of tumor cells and low estrogen-dependent ER activity in HER2-positive tumors.

337 (PB117) - Identification and molecular characterization of invasive lobular breast cancer models in a panel of 180 breast XPDX models

Background: Invasive lobular and ductal breast tumors have distinct histologies and clinical presentation. Invasive lobular cancer (ILC) characteristics include somatic alterations in the CDH1 gene leading to loss of E-cadherin expression, low HER2 expression, frequent loss of PTEN, increased AKT expression and mutations in PIK3CA, TBX3 and FOXA1 genes. Invasive ductal cancers (IDC) express E-cadherin and more often include HER2+ cancers and ESR1 mutations. The XenoSTART Patient-Derived Xenograft (XPDX) breast cancer platform includes over 180 models spanning all subtypes characterized with immunohistochemistry (IHC) including ERα, PR and HER2 protein levels, genomic and transcriptomic sequencing and in in vivo drug sensitivity. To further annotate our platform, we classified each breast XPDX model as ILC or IDC based on clinical history, E-cadherin staining, presence of variants in target genes and HER2 expression or amplification. Methods: XPDX models were established and validated as previously described and further characterized using IHC to determine E-cadherin protein levels (Abcam a-Rabbit ab15148). Analysis was performed on WES to identify variants in CDH1, PIK3CA, TBX3, FOXA1 and PTEN. RNAseq data was used to determine expression levels of AKT and HER2. Clinical histology data was also used for ILC versus IDC classification. Resulting models classified as ILC by our analysis were histologically confirmed by a clinical pathologist. Results: 180 breast models were examined by IHC and 25/180 (14%) lacked E-cadherin staining. Six of these 25 models (24%) reported CDH1 variants including deletions, frameshifts and point mutations. PIK3CA mutations, most notably H1047R, were reported in 5/25 models and FOXA1 (Q209E) was co-mutated in 2 of these models (ST2167 and ST2167/TDR). High AKT1 expression was reported in 5/25 models. While most E-cadherin-negative models were from clinically HER2 negative patients, 4/25 were from clinically HER2+ patients and corresponding models retained HER2 staining and amplification. 3/25 models were clinically classified as metaplastic and did not contain any target gene variant. Conclusion: We screened 180 models in our XPDX breast cancer platform and identified at least twenty-two ILC models based on E-cadherin staining, genomics and clinical histology, and confirmed heterogeneity of this subtype of breast cancer. Additonal analysis is underway to compare sensitivity of chemotherapy and targeted agents in ILC and IDC-classified breast models. No conflict of interest.

Treatment patterns and health care resource use of patients with metastatic breast cancer with HER2-low expression: A cancer registry-linked insurance claims study.

399 Background: Metastatic breast cancer (mBC) expresses varied levels of HER2 protein. While HER2+ mBC patients are treated with anti-HER2 therapies, those with lower levels of HER2 expression [HER2-low; immunohistochemistry (IHC) 1+, IHC 2+ with In Situ Hybridization (ISH) -] are currently classified as HER2-negative and are not treated with anti-HER2 therapies. This study aims to estimate population-based prevalence of HER2-low expression among mBC patients and assess healthcare resource utilization and breast cancer specific mortality rates, comparing HER2-low and HER2 IHC 0 patients. Methods: This was a retrospective population-based cohort study of women diagnosed with de novo mBC in North Carolina from 2010-2016, using a multi-payer linkage of insurance claims to state cancer registry data. We examined demographic and clinical characteristics, inpatient, emergency department (ED), and outpatient infusion visits over 12 months and lines of systemic therapy over 24 months by HER2 status and hormone receptor (HR) status. We estimated rates of hospitalization, emergency department (ED) and outpatient infusion visits per 1000-person days as well as hazard ratios for 3-year breast cancer specific mortality. Results: The cohort included 635 mBC patients; 53% (n = 337) met HER2-low criteria and 23.1% (n = 147) were HER2 IHC 0. Compared to HER2 IHC 0 patients, more HER2-low patients had hormone receptor (HR)+ disease (82% vs. 71%, p = 0.008) and were Black (26% vs. 16%, p = 0.029). HER2-low patients also had slightly higher rates of hospitalization (21.4 vs. 18.9, p = 0.003) and ED visits (2.4 vs. 1.8, p = 0.015), but similar rates of outpatient infusion visits (15.7 vs. 16.0, p = 0.684) compared to HER2 IHC 0. There were no differences in mortality due to mBC, comparing HER2 IHC 0 and HER2-low patients (HR: 0.90, 95% CI: 0.68-1.20, p = 0.483). Among HER2-low/HR+ patients, the most common regimens in first line (1L) included endocrine monotherapy (46.6%), chemotherapy (combination or monotherapy) (32.9%), and endocrine therapy with CDK4/6 inhibitors (18.8%). Similar patterns were seen in the second line (2L) with endocrine monotherapy (63.5%), chemotherapy (combination or monotherapy) (25.9%), and endocrine therapy with CDK4/6 inhibitors (9.5%). Most HER2-low/HR- patients received chemotherapy in 1L (88.9%) and 2L (90.9%). Conclusions: About 1 in 2 mBC patients met criteria to be classified as HER2-low. Overall, HER2-low patients experienced slightly higher rates of healthcare utilization than HER2 IHC 0 patients but had similar outcomes. Among HER2-low patients, treatment patterns by HR status were consistent with the standard of care for HER2 IHC 0 patients. There may be potential to improve outcomes among HER-low patients with anti-HER2 therapies demonstrating efficacy across levels of HER2 expression.

ABL kinases regulate translation in HER2+ cells through Y-box-binding protein 1 to facilitate colonization of the brain.

SUMMARYPatients with human epidermal growth factor receptor 2-positive (HER2+/ERBB2) breast cancer often present with brain metastasis. HER2-targeted therapies have not been successful to treat brain metastases in part due to poor blood-brain barrier (BBB) penetrance and emergence of resistance. Here, we report that Abelson (ABL) kinase allosteric inhibitors improve overall survival and impair HER2+ brain metastatic outgrowth in vivo. Mechanistically, ABL kinases phosphorylate the RNA-binding protein Y-box-binding protein 1 (YB-1). ABL kinase inhibition disrupts binding of YB-1 to the ERBB2 mRNA and impairs translation, leading to a profound decrease in HER2 protein levels. ABL-dependent tyrosine phosphorylation of YB-1 promotes HER2 translation. Notably, loss of YB-1 inhibits brain metastatic outgrowth and impairs expression of a subset of ABL-dependent brain metastatic targets. These data support a role for ABL kinases in the translational regulation of brain metastatic targets through YB-1 and offer a therapeutic target for HER2+ brain metastasis patients.

Synergistic Cytotoxic and Antimigratory Effect of Brazilein and Doxorubicin on HER2-Overexpressing Cells.

The present research aims to report cytotoxic and antimigratory activities of the oxidized form of brazilin, i.e., brazilein, and the effects of the combination of brazilein-doxorubicin on MCF-7/HER2 cells. The MTT assay was conducted to test the cytotoxic activity, while flow cytometry with PI and PI-annexin V staining were respectively performed for cell cycle and apoptosis analyses. Migration and invasion analyses were assessed via Boyden chamber assay, while HER2, Rac1, p120, MMP2, and MMP9 protein levels were determined by immunoblotting and gelatin zymography. Molecular docking of ligands with HER2, Src, PI3Kα, PI3KΔ, and PI3Kγ proteins was evaluated using MOE 2010. The MTT assay showed that the IC50 value of brazilein against MCF-7/HER2 cells was 51 ± 2.1 µM. Moreover, brazilein and its combination with doxorubicin-induced G2/M accumulation and apoptosis. Combination of brazilein-doxorubicin inhibited cell migration and tended to decrease HER2, Rac1, p120, MMP2, and MMP9 protein expression levels. Based on our molecular docking study, the docking score of brazilein with PI3Kγ is comparable to that of the native ligand. Taken together, a combination of brazilein-doxorubicin exhibited synergistic cytotoxic and antimigratory effects on MCF-7/HER2 cells.<br />.

Abstract 5661: HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers

Abstract HER2 is amplified in about 20% of breast cancers. HER3 is as essential as HER2 for maintaining cell viability in HER2+ breast cancer cells. It is known that inhibition of HER2 tyrosine kinase activity results in upregulation of HER3 transcription and phosphorylation. We sought to identify HER3 binding partners upon pharmacological inhibition of HER2 using neratinib. We immunoprecipitated HER3 using a HER3 antibody from BT474 cells treated ± neratinib. Fmass spectrometry experiments identified non-muscle myosin IIA (NMIIA) increased upon inhibition of HER2 with neratinib and decreased under DMSO control treatment from HER3 immunoprecipitates. To validate the presence of NMIIA, we performed immunoprecipitation experiments in BT474 and MDA-MB-453 cells using a HER3 antibody. Immunoblots showed increased NMIIA levels upon treatment with 200nM neratinib for 24 hours in both cell lines. Myosin heavy chain 9 (MYH9) gene encodes a protein called non-muscle myosin of class II, isoform A (NMIIA). It localizes to actin stress fibers and has been implicated in many cell functions. To confirm the interaction between HER3 and NMIIA we immunoprecipitated NMIIA from BT474 and MDA-MB-453 cells using a NMIIA antibody. Immunoblots indicated that HER3 levels were increased upon inhibition of HER2 with 200 nM neratinib for 24 hours. We next examined overall survival of primary breast cancer patients who have high gene expression for MYH9 from the METABRIC cohort. We observed that patients with high levels of MYH9 have a statistically significant worse overall survival versus patients with low levels of MYH9. Furthermore, we evaluated the overall levels of HER3 and MYH9 mRNA and protein upon treatment with neratinib in BT474 and MDA-MB-453 whole cell lysates. RT-qPCR and immunoblots showed increased HER3 and NM-IIA mRNA and protein levels upon neratinib treatment for 24 hours We examined the affect NMIIA loss has on HER3 signaling. Transduced MDA-MB-453 and BT474 cells with shMYH9 and doxycycline induction demonstrate a reduction in HER3 protein levels compared to cells transduced with a control sequence and doxycycline induction. We observed a concomitant reduction in P-HER3 (Y1289), downstream P-Akt (T308), and P-Erk1/2. In addition, NM-IIA knockdown suppresses cells growth, proliferation, migration, and invasion. In conclusion, there is a bidirectional relationship between NMIIA and HER signaling in HER2+ breast cancer cells. HER2 inhibition increases NMIIA and NMIIA promotes HER3 expression. Loss of NMIIA reduces HER3 protein and concomitant reductions in PI3K/Akt and MAPK signaling. Loss of NMIIA in combination with HER2 inhibition results in reduction in HER2+ breast cancer cell proliferation, growth on matrigel, migration, and invasion. Studies are ongoing to decipher the mechanisms for the bidirectional relationship between NMIIA and HER signaling. Citation Format: Samar M. Alanazi, Rosalin Mishra, Hima Patel, Mary K. Kilroy, Joan T. Garrett. HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5661.

Multiomic Profiling Identified EGF Receptor Signaling as a Potential Inhibitor of Type I Interferon Response in Models of Oncolytic Therapy by Vesicular Stomatitis Virus.

Cancer cell lines responded differentially to type I interferon treatment in models of oncolytic therapy using vesicular stomatitis virus (VSV). Two opposite cases were considered in this study, glioblastoma DBTRG-05MG and osteosarcoma HOS cell lines exhibiting resistance and sensitivity to VSV after the treatment, respectively. Type I interferon responses were compared for these cell lines by integrative analysis of the transcriptome, proteome, and RNA editome to identify molecular factors determining differential effects observed. Adenosine-to-inosine RNA editing was equally induced in both cell lines. However, transcriptome analysis showed that the number of differentially expressed genes was much higher in DBTRG-05MG with a specific enrichment in inflammatory proteins. Further, it was found that two genes, EGFR and HER2, were overexpressed in HOS cells compared with DBTRG-05MG, supporting recent reports that EGF receptor signaling attenuates interferon responses via HER2 co-receptor activity. Accordingly, combined treatment of cells with EGF receptor inhibitors such as gefitinib and type I interferon increases the resistance of sensitive cell lines to VSV. Moreover, sensitive cell lines had increased levels of HER2 protein compared with non-sensitive DBTRG-05MG. Presumably, the level of this protein expression in tumor cells might be a predictive biomarker of their resistance to oncolytic viral therapy.

Abstract TF2-2: Heterogeneity in treatment outcomes for HER2+ breast cancer - Clinical management strategies

Abstract HER2-positive breast cancers are complex tumors that are clinically and biologically heterogeneous. Even within HER2+ breast cancer, there are different molecular subtypes, mutational profiles, levels of HER2 protein, and immune infiltration. Additionally, HER2 may not be expressed homogeneously among all the cancer cells in the same tumor, some tumors may have low HER2 expression, while other tumors evolve during treatment. These molecular differences help explain why patients do not benefit to the same extent to HER2-directed therapies. We will review data regarding outcomes for tumors with known tumor heterogeneity, hormone receptor positivity, HER2-low positivity, and cases where brain metastases may be present. We will discuss optimal clinical management in the modern era with multiple HER2-directed therapies, including next-generation antibody-drug conjugates. Future strategies to tailor therapy to the individual patient will need to not just factor in anatomic tumor burden, but also key molecular features. Citation Format: S Tolaney. Heterogeneity in treatment outcomes for HER2+ breast cancer - Clinical management strategies [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr TF2-2.

Abstract P5-13-14: Antitumor activity of patritumab deruxtecan (HER3-DXd), a HER3-directed antibody drug conjugate (ADC) across a diverse panel of breast cancer (BC) patient-derived xenografts (PDXs)

Abstract Background: HER3 is overexpressed in 30-50% of breast cancers and has been associated with poor prognosis. Patritumab deruxtecan (HER3-DXd; U3-1402) is a HER3-directed ADC with a potent topoisomerase I (TOP1) inhibitor payload. A phase 1/2 study of HER3-DXd (NCT02980341) demonstrated promising antitumor activity in hormone receptor positive (HR+) metastatic BC patients with clinical activity observed across baseline levels of HER3 protein or mRNA expression. A window of opportunity clinical trial is currently ongoing to evaluate the biological activity of HER3-DXd in patients with treatment naïve BC according to HER3 mRNA/protein expression levels (NCT04610528). Here, we aimed to describe the activity of HER3-DXd in PDX models to identify robust biomarkers of response. Methods: The antitumor activity of HER3-DXd was assessed in 21 BC PDX models (14 HR+ and 7 triple negative). HER3-DXd sensitivity was established as a complete response that lasted longer than 120 days, following 4 weekly doses of 10 mg/kg. HER3-DXd antitumor activity was compared to the antitumor activity of irinotecan (50 mg/kg dosed once weekly), which was evaluated according to modified RECIST criteria in a subset of 14 BC PDX models. PDX models of acquired resistance to HER3-DXd were generated by repeated treatment cycles. Pharmacodynamic (PD) experiments were conducted by collecting tumor samples from PDXs after a single dose of HER3-DXd or irinotecan. Baseline HER3 expression was assessed by immunohistochemistry (IHC) and Western blot. mRNA expression of 72 genes including ERBB3 and genes from the PAM50 signature were measured using the nCounter platform. Two-class unpaired significance analysis of microarrays (SAM), using a false-discovery rate&amp;lt;5%, identified differential gene expression across response groups. Genetic alterations harbored by PDX models were determined using the MSK-IMPACTTM targeted exome panel. Quantification of proliferation (% of Ki67-positive cells) and of DNA damage during the S-phase of the cell cycle (γH2AX nuclear foci in geminin-positive cells) was evaluated in untreated/treated PD samples by IHC or immunofluorescence (IF), respectively. Western blot was used to assess HER3-pathway downmodulation and induction of apoptosis. Results: Eight out of 21 (38%) PDXs were highly sensitive to HER3-DXd, and in 5/14 (36%) models HER3-DXd showed a superior antitumor activity when compared to irinotecan. We observed an enrichment of basal-like models amongst the non-relapsed PDXs, compared to the relapsed ones (6/8 (75%) vs. 3/13 (20%), p=0.0195). Baseline levels of HER3/ERBB3 were not associated with treatment response and a model of acquired-resistance did not exhibit a reduction in baseline HER3 expression. Interestingly, relapsed models showed increased expression of genes related with chemotherapy-resistance (MDM2, NAT1, MAPT, GRB7, BCL-2). Mechanistically, treatment with HER3-DXd did not reduce the level of Ki67-proliferating cells but resulted in a significantly higher induction of S-phase DNA damage measured as γH2AX nuclear foci in non-relapsed models, compared to relapsed ones. This was accompanied by a reduction of HER3 protein levels and downmodulation of pERK1/2 T202/Y204, along with activation of PARP cleavage. Conclusions: HER3-DXd exerts a potent antitumor response in BC PDXs, independently of baseline HER3/ERBB3 levels, in line with the clinical data of NCT02980341. Basal-like tumors were more sensitive to HER3-DXd than luminal B models. Mechanistically, our data suggests that treatment with HER3-DXd results in parallel HER3/ERK signaling downmodulation and induction of S-phase DNA damage, resulting in tumor cell death. Citation Format: Andreu Òdena, Laia Monserrat, Fara Brasó-Maristany, Marta Guzmán, Judit Grueso, Olga Rodríguez, Maurizio Scaltriti, Sarat Chandarlapaty, Yang Qiu, Kumiko Koyama, Mafalda Oliveira, Aleix Prat, Violeta Serra. Antitumor activity of patritumab deruxtecan (HER3-DXd), a HER3-directed antibody drug conjugate (ADC) across a diverse panel of breast cancer (BC) patient-derived xenografts (PDXs) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-14.

A Novel HER2-Selective Kinase Inhibitor Is Effective in HER2 Mutant and Amplified Non-Small Cell Lung Cancer.

This study describes unique mechanisms of action of a new mutant-selective HER2 kinase inhibitor that reduces both kinase activity and protein levels of HER2 in lung cancer.

Molecular and cellular evidence for hsa-miR-1254 suppressor effect against HER2 signaling in breast cancer.

HER2 signaling upregulation is a hallmark of breast cancer which is the second cause of cancer-related death in women. Here, we were looking for the candidate microRNAs which is capable of regulating the HER2 receptor and the genes of its downstream. To this aim, preliminary bioinformatics analysis introduced hsa-miR-1254 (miR-1254) as a potential common regulator of HER2, HER3, PIK3R2, and AKT1 genes. Then, reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analysis indicated a lower expression level of miR-1254 in breast cancer specimens, compared to their normal pairs. Furthermore, overexpression of miR-1254 resulted in HER2, HER3, PIK3R2, and AKT1 genes downregulation, detected by RT-qPCR and confirmed by western blot analysis and enzyme-linked immunosorbent assay test against AKT1, BAX, FADD, and HER2 protein levels in SKBR3 cells. Dual-luciferase assay also supported direct interaction of miR-1254 with MREs within 3' untranslated region sequences of HER2, HER3, PIK3R2, and AKT1 target genes. Overexpression of miR-1254 in SKBR3 cells was followed by increased BAX/BCL2 expression ratio, detected by RT-qPCR, and increased proportion of G1 cell population, detected by flow cytometry. Corroborated by cell cycle analysis, MTT, Annexin V-FITC, and Live-Dead cell staining assays, overexpression of miR-1254 in MDA-MB-231 cells showed opposing results following the overexpression of miR-1254. Taken together, results indicated that miR-1254 acts as cell-type-specific tumor suppressor that targets HER2, HER3, PIK3R2, and AKT1 transcripts. These results suggest miR-1254 as a potential therapeutic candidate for breast cancer subtypes.

Cancer-associated mutations in the p85α N-terminal SH2 domain activate a spectrum of receptor tyrosine kinases

The phosphoinositide 3-kinase regulatory subunit p85α is a key regulator of kinase signaling and is frequently mutated in cancers. In the present study, we showed that in addition to weakening the inhibitory interaction between p85α and p110α, a group of driver mutations in the p85α N-terminal SH2 domain activated EGFR, HER2, HER3, c-Met, and IGF-1R in a p110α-independent manner. Cancer cells expressing these mutations exhibited the activation of p110α and the AKT pathway. Interestingly, the activation of EGFR, HER2, and c-Met was attributed to the ability of driver mutations to inhibit HER3 ubiquitination and degradation. The resulting increase in HER3 protein levels promoted its heterodimerization with EGFR, HER2, and c-Met, as well as the allosteric activation of these dimerized partners; however, HER3 silencing abolished this transactivation. Accordingly, inhibitors of either AKT or the HER family reduced the oncogenicity of driver mutations. The combination of these inhibitors resulted in marked synergy. Taken together, our findings provide mechanistic insights and suggest therapeutic strategies targeting a class of recurrent p85α mutations.

Abstract 1401: MYC mediates retinoic acid resistance by suppressing cellular retinoic acid-binding protein (CRABP2) transcription in HER2-enriched breast cancers

Abstract Introduction: HER2-positive (HER2+) breast cancers express high levels of the growth-promoting HER2 protein. The development of a targeted drug, trastuzumab, has greatly improved clinical outcome for HER2+ patients. However, intrinsic and acquired resistance to trastuzumab are common. Clearly, additional therapeutic interventions are required for the clinical management of HER2-enriched breast cancers. Retinoic acid (RA) is a vital signaling molecule that regulates multiple biological processes, including cell proliferation, differentiation, and death. RA has been successfully used in the treatment of acute promyelocytic leukemia. RA also holds promise for the treatment of solid cancers like breast cancer. RA action is mediated through transcription factor RARα and the RA binding protein CRABP2, which deliver RA to RARα in the nucleus. The HER2 gene (ERBB2) is frequently co-amplified with the gene encoding RARα, a key determinant of RA sensitivity. It seems surprising, therefore, that HER2+ breast cancers are refractory to RA treatment. MYC is an oncogene that inhibits RARα activity in leukemia cells. Importantly, MYC is preferentially amplified and overexpressed in HER2+ breast cancers. My research aims to elucidate the mechanism underlying RA resistance in HER2+ breast cancers, with a special focus on the role of MYC. Hypothesis: MYC attenuates RA action by inhibiting the CRABP2-RARα pathway. Results: RNA levels of MYC are negatively correlated with CRABP2 RNA levels. Depletion of MYC upregulates CRABP2 at both the RNA and protein levels. Furthermore, we found that MYC binds to the CRABP2 promoter region suggesting that MYC may directly suppress CRABP2 gene transcription activity. Our results show that ectopic expression of MYC inhibits, whereas depletion of MYC activates RAR activity. Consistently, ectopic expression of MYC increases RA resistance, whereas depletion of MYC sensitizes cells to RA treatment. When CRABP2 is depleted along with MYC-knockdown, cell proliferation is rescued, suggesting that MYC mediates RA resistance, at least partially through downregulation of CRABP2. We also found that RA treatment enhances trastuzumab responsiveness in HER2+ breast cancer cells. Significance: This study sheds light on the role and mechanism of MYC in governing RA resistance in HER2+ breast cancer cells. Our results support the use of RA and trastuzumab for the treatment of subsets of patients with HER2+/low MYC breast cancers. Citation Format: Won-Shik Choi, Rong-Zong Liu, Roseline Godbout. MYC mediates retinoic acid resistance by suppressing cellular retinoic acid-binding protein (CRABP2) transcription in HER2-enriched breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1401.

Abstract SP139: HER2 targeted therapy: Determinants of response and mechanisms of resistance

Abstract HER2-positive (+) breast cancer (BC), accounting for 15-20% of all BCs, is characterized by overexpression, mostly via gene amplification, of HER2. HER2 is a key member of the HER family of 4 tyrosine kinase receptors. Multiple clinically available HER2-targeted therapies, including monoclonal antibodies, tyrosine kinase inhibitors (TKIs), and antibody-drug conjugates have revolutionized the outcome of patients with HER2+ BC. Despite these effective therapies, intrinsic and acquired resistance still occurs, posing a major challenge in the clinical management of this disease. A better understanding of the determinants of response and mechanisms of resistance may help develop personalized treatment approaches and new strategies to overcome resistance. Tumors that are truly addicted to HER2, clinically reflected especially under chemotherapy-sparing HER2-targeted therapy regimens, are associated with high and homogeneous levels of HER2 gene amplification, protein, and activity. Even in these HER2-addicted tumors, the efficacy of anti-HER2 therapy can be jeopardized by deregulations in the downstream PI3K pathway (e.g., PIK3CA mutations), which can lead to constitutive activation of the PI3K/AKT pathway and resistance. Given the functional redundancy of signaling from multiple HER receptor dimers and compensatory signaling within the pathway, dual anti-HER2 therapy has proven superior to single agents in achieving a more comprehensive blockade of the entire HER receptor layer and in anti-tumor efficacy. Further, in the HER2+ tumors that co-express ER, an unblocked, re-expressed and/or reactivated ER signaling can provide alternative proliferative and survival signals to evade sustained HER2 blockade, thus underscoring the need for concurrent blockade of HER2 and ER signaling. Nevertheless, effective inhibition of HER2 might prove challenging in some cases due to molecular masking of the HER receptors (e.g., mucins) or due to the de novo presence or acquisition of genetic, epigenetic or post-translational alterations in HER2 itself, including activating HER2 mutations (e.g., L755S), and p95HER2. We recently reported that acquired resistance to HER2-targeted therapy, especially TKIs, is mediated by the common HER2 L755S mutation, the clinical importance of which is underscored by the observation that this and other HER mutations are further enriched in the metastatic lesions compared to primary HER2+ tumors. On the other hand, when HER2 does remain effectively inhibited under potent HER2-targeted therapy, resistance can arise due to the upregulation of alternative escape pathways that transmit proliferative stimuli. These include activation of other receptor tyrosine kinases (e.g., AXL, FGFR), other downstream/intracellular signaling (e.g., SRC, YES1), or metabolic pathways (e.g., FASN and mevalonate pathways). Our recent data suggest that the mevalonate pathway offers an escape mechanism by providing alternative signaling through the YAP/TAZ-mTORC1-survivin axis to activate a transcriptional program that promotes resistant cell proliferation and survival, which can be overcome using inhibitors of mevalonate pathway (e.g., statins). Importantly, activation of the key cell cycle regulator cyclin D1/CDK4 complex has been shown to mediate resistance to HER2-targeted therapy and that CDK4/6 inhibitors, at least partly by also inhibiting mTORC1 activity, can overcome this resistance. Finally, the role of tumor microenvironment, including host immune components (e.g., TILs) and extracellular matrix components signaling via integrins, have been shown to play a role in modulating tumor response to treatment and in resistance. Together, these findings suggest new strategies to enhance sensitivity and overcome resistance to HER2-targeted therapy, some of which are already under clinical development. Citation Format: R Schiff, J Veeraraghavan, C De Angelis, C Osborne, MF Rimawi. HER2 targeted therapy: Determinants of response and mechanisms of resistance [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP139.

Abstract PS4-22: Prevalence of HER2-low and HER2-zero subgroups and correlation with response to neoadjuvant chemotherapy (NACT) in patients with HER2-negative breast cancer

Abstract INTRODUCTION Current definition of HER2-positive BC follows ASCO/CAP guidelines using immunohistochemistry (IHC) and/or in situ hybridization (ISH)-based techniques. However, HER2 expression can be variable in cells that lack ERBB2 amplification. For example, HER2-negative tumors can express some level of HER2 protein by IHC (i.e. 1+ or 2+ and a negative ISH result) and are identified as HER2-low. Others have no expression and are considered HER2-zero. Innovative therapies have shown promising activity in patients in HER2-low BC. The aim of this study is to evaluate the association of HER2-low and HER2-zero status with response to NACT in HER2-negative BC. METHODS Retrospective cohort of patients with HER2-negative BC treated with NACT in four institutions in Brazil. Protocols of diagnosis, treatment and follow-up were standardized and based on international guidelines. Tumors with HER2 IHC score 0 were classified as HER2-zero whereas tumors with HER2 score 1+ and those with HER2 score 2+ with FISH-negative were classified as HER2-low. Patients were treated with anthracycline- and taxane- based chemotherapy. The following clinicopathological data were evaluated, when available: age, ER, Ki67, tumor size, lymph node (LN) status and response to NACT according to pCR status and residual cancer burden (RCB) index. Primary objective was to evaluate the prevalences and compare pCR rates among HER2-zero and HER2-low cases. Secondary objectives were to perform the same comparison within the HR-positive (HR+) and HR-negative subgroups. Pearsons chi squared tests were performed and a p value of &amp;lt;0.05 was considered statistically significant. RESULTS 331 patients were included in this analysis. 63% were HR+and 37% were TNBC. 50% were HER2-zero and 50% HER-low (36% HER2 IHC 1+ and 14% HER2 IHC 2+/FISH-negative). Median age, initial tumor size, clinical LN status and Ki67 expression were similar among HER2-zero and HER2-low subgroups. In HR+ tumors, 42% (86/207) were HER2-zero and 58% (121/207) were HER2-low. In TNBC, 63% (78/124) were HER2-zero and 37% (46/124) were HER2-low (p&amp;lt;0.001, Pearsons chi squared test). The pCR rate was 26% (85/331) in the entire cohort. As expected, there was a higher rate of pCR in TNBC vs HR+ (50% vs 11%, p&amp;lt;0.001). We found a statistically significant difference in the pCR rates when comparing the HER2-zero versus HER2-low subgroups (31% vs 20%, p=0.03). However, this difference is mostly related to an imbalance between groups (HER2-zero subgroups had a higher proportion of TNBC). Among HR+ tumors, there was no difference in the pCR rates between HER2-zero and HER2-low subgroups (8% vs 13%, p=0.35). In TNBC, we identified an interesting but non-statistically significant difference in pCR in HER2-zero vs. HER2-low tumors (56% vs. 39%, p=0.09). In the TNBC cohort we identified a non-statistically significant difference in RCB 0-I in HER2-zero vs. HER2-low tumors (p=0.06). With a 30 month median follow-up, PFS and OS data are immature. CONCLUSION The distribution of HER2-zero and HER2-low cases is different in HR+ and TNBC. HER2-low is more frequent in HR+ and HER2-zero in TNBC. We identified a higher pCR rate in HER2-zero compared to HER2-low tumors, even though this difference is associated with an imbalance between the two groups. Still, we identified a trend to higher pCR rate in HER2-zero compared to HER2-low tumors even within the TNBC subgroup. Identification of HER2-low and HER2-zero tumors may have clinical implications that should be further explored. Citation Format: Tomas Reinert, Guilherme Parisotto Sartori, Alessandra AB Souza, Rodrigo Pellegrini, Mahira L Rosa, Nathalia Rossatto, Guilherme P Coelho, Isnard E Litvin, Felipe Zerwes, Eduardo Millen, Francisco P Cavalcante, Antonio L Frasson, Marcia S Graudenz, Carlos H Barrios. Prevalence of HER2-low and HER2-zero subgroups and correlation with response to neoadjuvant chemotherapy (NACT) in patients with HER2-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-22.

Abstract PS10-08: Tucatinib potentiates the activity of the antibody-drug conjugate T-DM1 in preclinical models of HER2-positive breast cancer

Abstract Background: Tucatinib is an orally administered, reversible, highly specific HER2 tyrosine kinase inhibitor recently approved by the FDA in combination with trastuzumab and capecitabine for adult patients with advanced unresectable or metastatic HER2-positive breast cancer (mBC), including patients with brain metastases, that have failed at least one anti-HER2 regimen in the metastatic setting. In a phase IB clinical trial, tucatinib in combination with the HER2-targeted antibody-drug conjugate (ADC) ado-trastuzumab emtansine (T-DM1) was well tolerated and demonstrated activity in heavily pre-pretreated patients with HER2-positive mBC (NCT01983501; Borges VF et al., 2018). We previously presented preclinical data that tucatinib increases the activity of trastuzumab-derived ADCs in HER2-positive breast cancer models. Here, we provide mechanistic insight that tucatinib potentiates the activity of T-DM1 by modulating HER2 protein dynamics and facilitating increased cytotoxic maytansinoid drug delivery. Methods: To assess changes to HER2 protein levels upon treatment with tucatinib, HER2-amplified breast cancer cell lines were analyzed by Western blot and quantitative FACS (qFACS). To probe the dynamics of HER2 at the cell surface upon binding to antibody therapeutics, SK-BR-3 cells were incubated with fluorescently labeled trastuzumab to mark HER2 at the cell surface. Cells were imaged over 72 hours to observe the internalization of surface-bound antibody. Concurrent experiments were conducted with trastuzumab labeled with QF01, a quenched fluor which fluoresces only upon lysosomal processing and can serve as a proxy for antibody catabolism. To directly measure the rates of ADC catabolism, lysates were generated from BT-474 cells treated with T-DM1 in the presence or absence of tucatinib over a 72 hour time course, and were analyzed by mass spectrometry for the T-DM1 adduct, Lys-MCC-DM1. Results: In HER2-amplified breast cancer cell lines, treatment with tucatinib increased overall and cell membrane-localized HER2 levels. As demonstrated by internalization assays, tucatinib had an initial effect that increased the dwell time of HER2 at the cell surface of SK-BR-3 cells. At later timepoints, HER2 bound to trastuzumab was internalized and directed towards lysosomes. These data were supported by parallel intracellular Lys-MCC-DM1 measurements, which demonstrated increased concentration of the adduct when TDM-1 was administered in combination with tucatinib. These data provide a mechanistic rationale as to why the co-administration of tucatinib with T-DM1 in vitro was synergistic by isobologram analysis, and why the combination of tucatinib with T-DM1 was more effective in vivo than either single agent alone in BT-474 xenografts and in PDX models tested, producing a higher proportion of partial or complete tumor regressions. Conclusions: The described preclinical in vitro and in vivo data of simultaneous dual HER2 inhibition with tucatinib and T-DM1, along with the results of the phase IB/2 clinical trial demonstrating preliminary safety and efficacy of the combination, warrant further clinical development of tucatinib in combination with T-DM1. These results also support the evaluation of tucatinib in combination with other HER2-targeted ADCs in patients with HER2-positive mBC. Citation Format: Anita Kulukian, Janelle Taylor, Nishi Jain, Devra Olson, Margo Zaval, Robert Thurman, Shawna Hengel, Lauren Farr, Thomas Pires, Scott R. Peterson. Tucatinib potentiates the activity of the antibody-drug conjugate T-DM1 in preclinical models of HER2-positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-08.

3D Visualization of the Dynamic Bidirectional Talk Between ER/PR and Her2 Pathways.

Background: Extensive amounts of archived formalin fixed paraffin embedded (FFPE) human tumor tissues are the ultimate resource to investigate signaling network underlying tumorigenesis in human. Yet, their usage is severely limited for lacking of suitable protein techniques. In this study, a quantitative, objective, absolute, and high throughput immunoblot method, quantitative dot blot (QDB), was explored to address this issue by investigating the putative relationship between estrogen receptor (ER)/progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) pathways in breast cancer tumorigenesis. Methods: In this descriptive observational retrospective study, ER, PR, Her2, and Ki67 protein levels were measured absolutely and quantitatively in 852 FFPE breast cancer tissues using the QDB method. ER, PR, and Her2 levels were charted on the X, Y, and Z-axes to observe samples distribution in a 3D scatterplot. Results: A “seesaw” relationship between ER/PR and Her2 pathways was observed in ER–PR–Her2 space, characterized by the expression levels of these 3 proteins. Specimens with strong expressions of ER/PR proteins were found spreading along the ER/PR floor while those with strong Her2 expression were found wrapping around the Her2 axis. Those lacking strong expressions of all 3 proteins were found accumulating at the intersection of the ER, PR, and Her2 axes. Few specimens floated in the ER–PR–Her2 space to suggest the lack of co-expression of all 3 proteins simultaneously. Ki67 levels were found to be significantly reduced in specimens spreading in the ER–PR space. Conclusions: The unique distribution of specimens in ER–PR–Her2 space prior to any clinical intervention provided visual support of bidirectional talk between ER/PR and Her2 pathways in breast cancer specimens. Clinical interventions to suppress these 2 pathways alternatively warrant further exploration for breast cancer patients accordingly. Our study also demonstrated that the QDB method is an effective tool to analyze archived FFPE cancer specimens in biomedical research.

Details of human epidermal growth factor receptor 2 status in 454 cases of biliary tract cancer

Human epidermal growth factor receptor 2 (HER2)-targeted therapy has improved clinical outcomes in patients with HER2-positive breast and gastric cancers, although ineffective or recurrent cases are present. One reason for this is the heterogeneity of HER2 expression in cancer cells. The aim of this study was to investigate the clinicopathological characteristics and HER2 status of patients with biliary tract cancers (BTCs). We examined HER2 protein expression by immunohistochemistry, HER2 gene amplification by fluorescence in situ hybridization, and both HER2 protein and gene levels simultaneously by gene-protein assay. Samples were collected from 454 patients who underwent surgical resection for BTCs (110 intrahepatic cholangiocarcinomas [ICC], 67 perihilar extrahepatic cholangiocarcinomas [ECC-Bp], 119 distal extrahepatic cholangiocarcinomas [ECC-Bd], 80 gallbladder carcinomas [GBC], and 79 ampullary carcinomas [AVC]). HER2 status was assessed according to the guidelines for HER2 testing in gastroesophageal adenocarcinoma. HER2-positive status was detected in 14.5% of BTCs (3.7% of ICC, 3.0% of ECC-Bp, 18.5% of ECC-Bd, 31.3% of GBC, and 16.4% of AVC). Furthermore, HER2-positivity tended to correlate with low histological grade, tumor histology, and macroscopic features in certain tumors. HER2 heterogeneity was common and highly frequent (83%) in BTC cases. Reduced HER2 protein expression in the deeper invasive areas with simultaneous dedifferentiation was frequently observed in HER2-positive cancer cells. The findings of this study suggest that a large subgroup of HER2-positive BTC cases can be considered for HER2-targeted therapy. Moreover, the HER2 status in BTCs should be determined carefully using a sensitive approach toward larger cancer tissues.

Abstract 5192: EGFR inhibition enhances the cellular uptake and antitumor activity of the novel HER3 antibody drug conjugate U3-1402

Abstract Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have changed the treatment landscape for EGFR-mutant non-small cell lung cancers (NSCLC); however, most patients develop resistance over time. HER3 is a unique pseudokinase member of the ERBB family which functions through dimerization with other ERBB family members (EGFR and HER2) and has been difficult to target with conventional kinase inhibitor strategies. HER3 is frequently over expressed in EGFR-mutant NSCLC. U3-1402 is an antibody-drug conjugate (ADC) comprised of HER3-targeting antibody (patritumab) linked to a topoisomerase I inhibitor (DX-8951 derivative, or DXd). U3-1402 is currently in clinical development as a single agent in metastatic EGFR-mutant NSCLC (NCT03260491). We aimed to develop a preclinical strategy to enhance the efficacy of U3-1402. Pre-treatment with EGFR TKIs (gefitinib or osimertinib) increased HER3 membrane levels in six different EGFR-mutant cell lines by increasing both the amount of HER3 positive cells and the intensity of HER3 expression. Furthermore, using a proximity-ligation assay (PLA), we noted an increase in membrane EGFR:HER3 interaction following EGFR inhibitor treatment. HER3 protein levels and EGFR:HER3 interaction were similarly increased in four EGFR-mutant patient-derived xenograft (PDX) models following osimertinib treatment. HER3 expression peaked between 24 and 48 hours, and the increase in HER3 was regulated by both increased gene expression and in the level of protein stability. We further evaluated the biological consequences of increased HER3 expression on U3-1402 intake and activity. We used pH-sensitive pHrodo Red conjugate to label U3-1402 to monitor the intake of the ADC using Incucyte live cell imaging. In three EGFR-mutant cell lines, there was a 3- to 5-fold higher intake of U3-1402 over time in cell lines pre-treated with osimertinib, compared to cell lines without pre-treatment. Osimertinib pre-treatment also increased the uptake of U3-1402 in HER3-low expressing EGFR-mutant models. In contrast, osimertinib pre-treatment did not increase the uptake of a control IgG-ADC. In long-term in vitro cell growth assays, the combination of osimertinib and U3-1402 was superior to single agent U3-1402 in four EGFR-mutant cell lines. Preliminary in vivo studies demonstrated no significant toxicities as measured by mouse body weights and complete blood counts. In vivo experiments evaluating the antitumor efficacy of the combination of osimertinib and U3-1402 are currently underway. Our studies reveal that EGFR inhibitor treatment increased membrane expression of HER3 which was associated with enhanced internalization of U3-1402 in EGFR-mutant NSCLC. The combination of osimertinib and U3-1402 may be an effective treatment approach and should be evaluated in future clinical trials in patients with EGFR-mutant NSCLC. Citation Format: Heidi M. Haikala, Jens Köhler, Timothy Lopez, Pinar Eser, Man Xu, Channing Yu, Yoshinobu Shiose, Yang Qiu, Prafulla Gokhale, Pasi A. Jänne. EGFR inhibition enhances the cellular uptake and antitumor activity of the novel HER3 antibody drug conjugate U3-1402 [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 5192.