ErbB Receptors Research Articles

Neratinib is a TFEB and TFE3 activator that potentiates autophagy and unbalances energy metabolism in ERBB2+ breast cancer cells

Neratinib (NE) is an irreversible pan-ERBB tyrosine kinase inhibitor used to treat breast cancers (BCa) with amplification of the ERBB2/HER2/Neu gene or overexpression of the ERBB2 receptor. However, the mechanisms behind this process are not fully understood. Here we investigated the effects of NE on critical cell survival processes in ERBB2+ cancer cells. By kinome array analysis, we showed that NE time-dependently inhibited the phosphorylation of two distinct sets of kinases. The first set, including ERBB2 downstream signaling kinases such as ERK1/2, ATK, and AKT substrates, showed inhibition after 2 h of NE treatment. The second set, which comprised kinases involved in DNA damage response, displayed inhibition after 72 h. Flow cytometry analyses showed that NE induced G0/G1 cell cycle arrest and early apoptosis. By immunoblot, light and electron microscopy, we revealed that NE also transiently induced autophagy, mediated by increased expression levels and nuclear localization of TFEB and TFE3. Altered TFEB/TFE3 expression was accompanied by dysregulation of mitochondrial energy metabolism and dynamics, leading to a decrease in ATP production, glycolytic activity, and a transient downregulation of fission proteins. Increased TFEB and TFE3 expression was also observed in ERBB2-/ERBB1 + BCa cells, supporting that NE may act through other ERBB family members and/or other kinases. Overall, this study highlights NE as a potent activator of TFEB and TFE3, leading to the suppression of cancer cell survival through autophagy induction, cell cycle arrest, apoptosis, mitochondrial dysfunction and inhibition of DNA damage response.

The changes of HER3 expression in head and neck cancer patients treated with induction chemotherapy.

e18004 Background: Erb-b2 receptor tyrosine kinase 3 (HER3) is broadly expressed in various types of cancer and is associated with poor prognosis. Recent evidence supports HER3 plays a role in acquired resistance to chemotherapy. The change of HER3 expression by chemotherapy in head and neck squamous cell carcinoma (HNSCC) remains unclear. Methods: We evaluated 95 patients with locally advanced HNSCC who had received induction chemotherapy (ICT) between 2009 and 2019. Immunohistochemical staining for HER3 was performed using formalin-fixed paraffin-embedded surgical specimens and biopsy samples. H-score of membrane staining was evaluated. Results: Median age of patients was 64 years, and 85% were male. Primary locations included oropharynx (54.8%), hypopharynx (21.5%), oral cavity (17.2%), and salivary gland (6.5%). Mean H-score of HER3 in tumor tissues was 77.6 ± 7.9. H-score of HER3 in HPV-positive tumors was higher than HPV-negative tumors (p = 0.019), which was more pronounced in oropharynx and hypopharynx tumors. While, H-score of HER3 in adjacent normal tissues of these tumors did not significantly differ. Compared with the non-smokers, tumor tissues of current or ex-smokers demonstrated higher H-score of HER3 (p = 0.033), independent of tumor location. In contrast, in adjacent normal tissues, no significant differences of H-score of HER3 were observed depending on the smoking status. The H-score higher than 100 was defined as HER3-high group. Among oropharynx and hypopharynx tumors with positive correlation between H-score of HER3 and HPV status (n = 56), HER3-high group was significantly correlated with HPV positivity (p = 0.009) and good response to ICT (p = 0.035). Good clinical response was also strongly related to HPV-positivity (p = 0.001). In addition, there was a tendency for longer overall survival in HER3-high group than low HER3 expression group (93 vs. 70 months, p = 0.053). The paired tumor samples collected at pre- and post-ICT (n = 21) were analyzed. HER3 expressions in adjacent normal tissue were higher at post-ICT compared to pre-ICT (p = 0.003), and these results were more pronounced in the non-smoking group, but no differences were found according to HPV-status. Conclusions: Taken together, our results showed that H-score of HER3 was significantly higher in both HPV-positive and smoking group. Interestingly, high HER3 expression in oropharynx and hypopharynx tumors was associated with HPV positivity and good clinical response to ICT. Additional analysis of tumor immune microenvironment in tumor tissues are underway.

Genomics of ERBB2-Positive Breast Cancer in Young Women Before and After Exposure to Chemotherapy Plus Trastuzumab.

Erb-B2 receptor tyrosine kinase 2 (ERBB2)-positive breast cancer (BC) is particularly common in young women. Genomic features of ERBB2-positive tumors before and after chemotherapy and trastuzumab (chemo + H) have not been described in young women and are important for guiding study of therapeutic resistance in this population. From a large prospective cohort of women age 40 years or younger with BC, we identified patients with ERBB2-positive BC and tumor tissue available before and after chemo + H. Whole-exome sequencing (WES) was performed on each tumor and on germline DNA from blood. Tumor-normal pairs were analyzed for mutations and copy number (CN) changes. Twenty-two women had successful WES on samples from at least one time point; 12 of these had paired sequencing results from before and after chemo + H and 10 had successful sequencing from either time point. TP53 was the only significantly recurrently mutated gene in both pre- and post-treatment samples. MYC gene amplification was observed in four post-treatment tumors. Seven of 12 patients with paired samples showed acquired and/or clonally enriched alterations in cancer-related genes. One patient had an increased clonality putative activating mutation in ERBB2. Another patient acquired a clonal hotspot mutation in TP53. Other genomic changes acquired in post-treatment specimens included alterations in NOTCH2, STIL, PIK3CA, and GATA3. There was no significant change in median ERBB2 CN (20.3 v 22.6; Wilcoxon P = .79) between paired samples. ERBB2-positive BCs in young women displayed substantial genomic evolution after treatment with chemo + H. Approximately half of patients with paired samples demonstrated acquired and/or clonally enriched genomic changes in cancer genes. ERBB2 CN changes were uncommon. We identified several genes warranting exploration as potential mechanisms of resistance to therapy in this population.

Deriving breast cancer chemotherapy patterns from real-world data.

e13586 Background: Real World Data (RWD) collected during routine medical practice can help in clinical trial design, planning and execution. In addition, it can provide a compelling picture of safety of medical products, and account for all potential adverse events that can be encountered in routine clinical practice. However, raw drug information about chemotherapy in cancer care is not readily understandable in the format it is collected. Analytical techniques need to be applied to extract regimen information, which includes drugs, dosage, number of cycles and cycle length. Methods: A retrospective study was performed using data on 7,798 breast cancer patients from the TriNetX Network, a federated network of de-identified, HIPAA-compliant, health data from 21 healthcare organizations across North America as of May 2022. We investigated a method built on rule-based algorithm and clustering analysis to extract regimens and their patterns of administration and align them into lines of treatment (LOT). To derive drug administration patterns, we clustered administration time periods using three features: total number of drug administrations, median number of days between drug administrations, and standard deviation of the days between drug administrations. Results: The patterns of administration that correspond to the two most common regimens for Erb-B2 receptor tyrosine kinase 2 negative group (ERBB2-) stages 1, 2 and 3 are shown. We looked at patients with Hormone Receptor (HR+) positive and Triple Negative (TN) breast cancer. Results of our analysis were in a close agreement with NCCN Guidelines. However, regimen administration patterns varied. This information can be useful to characterize patients based on the adherence to the expected chemotherapy regimens. It can also provide meaningful insight into burden of illness, such that patients who have higher variability of the drug administration might be faced with clinical problems such as drug tolerability or side effects. The variability might also signify access to care challenges. Conclusions: Understanding regimen LOT and administration patterns is central to research based on RWD, but it can also be useful for clinical trial design, execution, as well as site selection and patient recruitment for clinical trials. [Table: see text]

HER3 expression status following systemic chemotherapy treatment in biliary tract cancers.

e16153 Background: Erb-b2 receptor tyrosine kinase 3 (HER3) is frequently overexpressed in a variety of cancers. Activation of HER3 signaling serves an important role in promoting cell proliferation and is associated with chemoresistance. Thus, there is interest in exploring HER3 status of tumors as a potential prognostic and/or therapeutic target. Prior studies have reported HER3 status in various cancers after treatment with chemotherapy (CT). However, HER3 expression profile in biliary tract cancer (BTC) remains unknown. Here, we evaluated HER3 status of patients with BTC post CT. Methods: The medical records of patients (pts) who had been initially diagnosed with BTC and treated at the National Cancer Center Hospital (Tokyo, Japan), between January 2010 and June 2020, were retrospectively analyzed. Pts whose post CT specimens were available were included in this study. Immunohistochemical staining for HER3 was performed using HER3/ErbB3 (D22C5) XP Rabbit mAb (Cell Signaling Technology) as a primary antibody. IHC scoring (0, 1+, 2+, 3+) of membranous staining intensity was performed according to HER2 IHC gastric scoring guideline. Results: A total of 19 pts with BTC, with a median age of 59 (range 44-79) years, had specimens available after post CT and were evaluated for HER3 expression status. HER3 status was 3+ in 10 pts, 2+ in 3 pts, 1+ in 4 pts, and 0 in 2 pts. The specimens were obtained from liver in 17 pts, bone in 1 pt, and omental lymph node in 1 pt. The histological type was adenocarcinoma in all pts, and HER3 status according to primary tumor type is shown in the table below. Fourteen pts were treated with gemcitabine (GEM) plus cisplatin (CDDP), 1 pt with GEM plus CDDP plus S-1, 1 pt with GEM plus S-1 as first-line CT for advanced BTC, and 3 pts with S-1 as an adjuvant treatment. Fifteen pts were biopsied after CT to participate in clinical trial. The remaining 4 obtained specimens after CT during primary surgery or surgery for metastases or for cholangitis. The median overall survival of pts with advanced BTC was 23.6 months in HER3 3+, 17.1 months in HER3 2+, 27.7 months in HER3 1+, and 24.6 months in HER3 0. Four pts had specimens available from pre and post CT, all of which were intrahepatic cholangiocarcinoma with the treatment of GEM plus CDDP. HER3 status was upregulated in 3 pts (1 pt 1+ to 2+, 2 pts 2+ to 3+) and downregulated in 1 pt (1+ to 0) at post CT. Conclusions: HER3 expression (1+, 2+, 3+) was observed in 89.5% of previously treated pts with BTC, and HER3 status was upregulated after CT in some pts. These results suggest that HER3 might be a potential therapeutic target in BTC post CT. [Table: see text]

Betacellulin regulates gap junction intercellular communication by inducing the phosphorylation of connexin 43 in human granulosa-lutein cells

BackgroundThe gap junction protein, connexin 43 (Cx43) is highly expressed in human granulosa-lutein (hGL) cells. The phosphorylation of certain amino acid residues in the Cx43 protein has been shown to be related to a decline in gap junction intercellular communication (GJIC), which subsequently affects oocyte meiotic resumption. As a member of the epidermal growth factor (EGF) family, betacellulin (BTC) mediates luteinizing hormone (LH)-induced oocyte maturation and cumulus cell expansion in mammalian follicles. Whether BTC can regulate Cx43 phosphorylation, which further reduces Cx43-coupled GJIC activity in hGL cells remains to be determined.MethodsImmortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing in vitro fertilization in an academic research center were used as the study models. The expression levels of Cx43 and phosphorylated Cx43 were examined following cell incubation with BTC at different time points. Several kinase inhibitors (sotrastaurin, AG1478, and U0126) and small interfering RNAs targeting EGF receptor (EGFR) and receptor tyrosine-protein kinase 4 (ErbB4) were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance.ResultsThe results showed that BTC induced the rapid phosphorylation of Cx43 at serine368 without altering the expression of Cx43 in primary and immortalized hGL cells. Additionally, using a dual inhibition approach (kinase inhibitors and siRNA-based expression knockdown), we demonstrated that this effect was mainly mediated by the EGFR but not the ErbB4 receptor. Furthermore, using a protein kinase C (PKC) kinase assay and a scrape-loading and dye transfer assay, we revealed that PKC signaling is the downstream signaling pathway that mediates the increase in Cx43 phosphorylation and subsequent decrease in GJIC activity in response to BTC treatment in hGL cells.ConclusionsBTC promptly induced the phosphorylation of connexin 43 at Ser368, leading to decreased GJIC activity in hGL cells. The BTC-induced cellular activities were most likely driven by the EGFR-mediated PKC-dependent signaling pathway. Our findings shed light on the detailed molecular mechanisms by which BTC regulates the process of oocyte meiotic resumption.

Scalp acupuncture improves disordered behavior in the valproic acid-induced rat model of autism: 头针改善丙戊酸钠诱导的孤独症大鼠行为学及其机制研究

ObjectivesTo investigate the underlying mechanisms of scalp acupuncture treatment (SAT) on autism spectrum disorder (ASD). MethodsThirty male Wistar rat pups that had been prenatally exposed to valproic acid sodium (VPA) were randomly divided into the VPA, VPA+acupoint, and VPA+ non-acupoint groups using the random number table method, with 10 rats in each group. Ten pups who had been prenatally exposed to saline were assigned to the control group (CG). There was no intervention in either the control or VPA groups. In the acupoint group, “Shenting (GV24),” bilateral “Benshen (GB13)” were manipulated. In the non-acupoint group, the area below the costal space was stimulated. Acupuncture stimulation lasted for 40 min, with manual twisting of the needles every 10 min, 5 days/week, with 2 days of rest per week, for a total duration of 4 weeks. After the corresponding treatments, behavioral tests (including the open field, social interaction, and Morris water maze tests) were performed to evaluate the therapeutic effects. RT-PCR and Western blotting were performed to detect the expression of neuregulin 1 (NRG1)/ErbB4. ResultsIn the open field test, the activity time spent in the central area in the VPA+acupoint group was significantly longer than that in the VPA group and VPA+ non-acupoint group (both P<0.05). The total length in the VPA+acupoint group was significantly longer than that in the VPA group (P<0.05). The number of bouts in the central area of the VPA+acupoint group was significantly higher than that of the VPA group (P<0.05). In session I of social interaction test, all experimental rats spent more time interacting with stranger 1 (all P<0.05). In session II, the CG and VPA+acupoint groups rats showed more interest in searching for new strangers, but the VPA+non-acupoint group spent more time interacting with stranger 1 than with stranger 2(all P<0.05). In the Morris water maze test, compared with the VPA group, the latency of the VPA+acupoint group was shorter (day 2, 3, 4, 5, P<0.05); compared with VPA+acupoint group, the latency of the VPA+non-acupoint group was longer (day 2, 4, P<0.05). The mean distance in the VPA+acupoint group was shorter than that in the VPA group (day 3, 5, P<0.05). The platform quadrant time of the VPA+non-acupoint group was significantly shorter than that of the VPA+acupoint group (P<0.05) (day 6). The VPA+acupoint group had more platform crossings than the VPA group (P<0.05), and the VPA+ non-acupoint group had fewer platform crossings than the VPA+acupoint group (P<0.05) (day 6). After SAT, the expression levels of NRG1 and ErbB4 proteins in the VPA+acupoint group were significantly increased than those in the VPA group (both P<0.05) in the medial prefrontal cortex (mPFC). The expression levels of NRG1 and ErbB4 proteins in VPA+non-acupoint group were significantly less than those in the VPA+acupoint group (both P<0.05) in the mPFC. After SAT, the expression levels of NRG1 and ErbB4 proteins in the VPA+acupoint group were significantly higher than those in the VPA group (both P<0.05) in the hippocampus. The expression levels of NRG1 and ErbB4 proteins in the VPA+non-acupoint group were significantly lower than those in the VPA+acupoint group (both P<0.05) in the hippocampus. After SAT, the expression levels of mRNA levels of NRG1-ErbB4 in the mPFC and hippocampus in VPA+acupoint group were significantly higher than those in the VPA group (all P<0.05). The mRNA expression levels of NRG1-ErbB4 in the mPFC and hippocampus in VPA+ non-acupoint group were significantly lower than those in the VPA+acupoint group (all P<0.05). ConclusionsOur results suggest that SAT can improve ASD-like behaviors in young rats in a VPA model of ASD. This may be related to its function in upregulating the expression of protein and gene of NRG1 and its receptor ErbB4 in the mPFC.

JK07 prevents atrial fibrosis and atrial fibrillation inducibility in a porcine DOCA model

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Geconcerteerde Onderzoeksactie (GOA) grant 499 of the University of Antwerp Industrieel OnderzoeksFonds/Strategisch Basisonderzoek 498 (IOF/SBO) research grant of the University of Antwerp Background Atrial fibrosis is a substrate of atrial fibrillation (AF), and higher burdens of fibrosis are associated with resistance to therapy and worse prognosis. Currently, no therapies exist that target atrial fibrosis. JK07 is a long-acting neuregulin-fusion protein that has been shown to decrease ventricular fibrosis in models of heart failure through selective stimulation of the ErbB4 receptor. Purpose To test the ability of JK07 to reduce atrial fibrosis and AF inducibility in minipig model of deoxycorticosterone acetate (DOCA, an aldosterone agonist) induced hypertension. Methods 18 Aachener minipigs were randomized into 3 groups: control (CTRL), DOCA + Vehicle (DOCA+VEH) and DOCA+JK07. The control group did not undergo a therapeutic intervention. To induce hypertension and atrial fibrosis, pellets releasing 10 mg/kg DOCA over a period of 60 days were implanted in minipigs in the DOCA+VEH and DOCA+JK07 groups. The DOCA-implanted animals underwent weekly treatment with JK07 (0.3 mg/kg; DOCA + JK07) or its vehicle (DOCA + VEH), starting at the day of implantation (total of 9 administrations). After 60 days, arterial blood pressure was measured invasively and a decapolar catheter was placed in the right atrium. AF inducibility was tested by performing 50 burst pacing episodes and quantified as the percentage of successful episodes where an AF run ≥ 5 sec could be induced after the burst, out of the 50 attempts. Atrial fibrosis was quantified using ImageJ software on Masson trichrome staining of left atrial specimens, isolated after euthanizing the animals. Results Mean arterial pressure was significantly higher in the DOCA+VEH (142 ± 10 mmHg) and DOCA+JK07 (132 ± 15 mmHg) groups than in the CTRL group (105 ± 8 mmHg, p&amp;lt;0.01), without an effect of JK07. AF inducibility in the DOCA+VEH group was significantly higher than in the CTRL group (66/250 vs. 9/300, p&amp;lt;0.001), and the DOCA + JK07 group (66/250 vs. 8/300, p&amp;lt;0.001). Likewise, the degree of atrial fibrosis was significantly higher in the DOCA+VEH group compared to CTRL (14.18 ± 1.81 vs. 8.30 ± 2.52, p&amp;lt;0.001) and compared to DOCA+JK07 (14.18 ± 1.81 vs. 10.65 ± 1.59, p=0.0049). Conclusions JK07 prevents atrial fibrosis and AF inducibility in a porcine DOCA model. The effect of JK07 is unrelated to effects on blood pressure, but probably related to reduced atrial fibrogenesis.

Lipid Metabolism Reprogramming and Trastuzumab Resistance in Breast Cancer Cell Lines Overexpressing the ERBB2 Membrane Receptor.

Trastuzumab (Tz), an antibody targeting ERBB2, has significantly improved the prognosis for breast cancer (BCa) patients with overexpression of the ERBB2 receptor. However, Tz resistance poses a challenge to patient outcomes. Numerous mechanisms have been suggested to contribute to Tz resistance, and this study aimed to uncover shared mechanisms in in vitro models of acquired BCa Tz resistance. Three widely used ERBB2+ BCa cell lines, adapted to grow in Tz, were examined. Despite investigating potential changes in phenotype, proliferation, and ERBB2 membrane expression in these Tz-resistant (Tz-R) cell lines compared to wild-type (wt) cells, no common alterations were discovered. Instead, high-resolution mass spectrometry analysis revealed a shared set of differentially expressed proteins (DEPs) in Tz-R versus wt cells. Bioinformatic analysis demonstrated that all three Tz-R cell models exhibited modulation of proteins associated with lipid metabolism, organophosphate biosynthesis, and macromolecule methylation. Ultrastructural examination corroborated the presence of altered lipid droplets in resistant cells. These findings strongly support the notion that intricate metabolic adaptations, including lipid metabolism, protein phosphorylation, and potentially chromatin remodeling, may contribute to Tz resistance. The detection of 10 common DEPs across all three Tz-resistant cell lines offers promising avenues for future therapeutic interventions, providing potential targets to overcome Tz resistance and potentially improve patient outcomes in ERBB2+ breast cancer.

EGFR and ERBB2 exon 20 insertion/duplication in advanced non-small cell lung cancer: genomic profiling and clinicopathologic features.

Exon 20 (ex20) in-frame insertions or duplications (ins/dup) in epidermal growth factor receptor (EGFR) and its analog erb-b2 receptor tyrosine kinase 2 (ERBB2) are each detected in 1.5% of non-small cell lung cancer (NSCLC). Unlike EGFR p.L858R or ex19 deletions, ex20 ins/dup is associated with de novo resistance to classic EGFR inhibitors, lack of response to immune checkpoint inhibitors, and poor prognosis. US Food and Drug Administration has approved mobocertinib and amivantamab for targeting tumors with this aberration, but the number of comprehensive studies on ex20 ins/dup NSCLC is limited. We identified 18 cases of NSCLCs with EGFR/ERBB2 ex20 ins/dup and correlated the findings with clinical and morphologic information including programed death-ligand 1 (PD-L1) expression. A total of 536 NSCLC cases tested at our institution between 2014 and 2023 were reviewed. A custom-designed 214-gene next-generation sequencing panel was used for detecting DNA variants, and the FusionPlex CTL panel (ArcherDx) was used for the detection of fusion transcripts from formalin-fixed, paraffin-embedded tissue. Immunohistochemistry (IHC)for PD-L1 was performed using 22C3 or E1L3N clones. Nine EGFR and nine ERBB2 ex20 ins/dup variants were identified from an equal number of men and women, 14 were non- or light smokers, and 15 had stage IV disease. All 18 cases were adenocarcinomas. Seven of the 11 cases with available primary tumors had acinar predominant pattern, two had lepidic predominant pattern, and the remainder had papillary (one case) and mucinous (one case) patterns. Ex20 ins/dup variants were heterogenous in-frame one to four amino acids spanning A767-V774 in EGFR and Y772-P780 in ERBB2 and were clustered in the loop following the C-helix and α C-helix. Twelve cases (67%) had co-existing TP53 variants. Copy number variation in CDK4 amplification was identified in one case. No fusion or microsatellite instability was identified in any case. PD-L1 was positive in two cases, low positive in four cases, and negative in 11 cases. NSCLCs harboring EGFR/ERBB2 ex20 ins/dup are rare and tend to be acinar predominant, negative for PD-L1, more frequent in non- or light smokers, and mutually exclusive with other driver mutations in NSCLC. The correlation of different EGFR/ERBB2 ex20 ins/dup variants and co-existing mutations with response to targeted therapy and the possibility of developing resistant mutations after mobocertinib treatment warrants further investigation.

Biased activation of the receptor tyrosine kinase HER2

HER2 belongs to the ErbB sub-family of receptor tyrosine kinases and regulates cellular proliferation and growth. Different from other ErbB receptors, HER2 has no known ligand. Activation occurs through heterodimerization with other ErbB receptors and their cognate ligands. This suggests several possible activation paths of HER2 with ligand-specific, differential response, which has so far remained unexplored. Using single-molecule tracking and the diffusion profile of HER2 as a proxy for activity, we measured the activation strength and temporal profile in live cells. We found that HER2 is strongly activated by EGFR-targeting ligands EGF and TGFα, yet with a distinguishable temporal fingerprint. The HER4-targeting ligands EREG and NRGβ1 showed weaker activation of HER2, a preference for EREG, and a delayed response to NRGβ1. Our results indicate a selective ligand response of HER2 that may serve as a regulatory element. Our experimental approach is easily transferable to other membrane receptors targeted by multiple ligands.Graphical abstract

Survival Outcomes in Patients With Hormone Receptor–Positive Metastatic Breast Cancer With Low or No ERBB2 Expression Treated With Targeted Therapies Plus Endocrine Therapy

Approximately 45% to 60% of hormone receptor (HR)-positive metastatic breast cancer (mBC) shows a low-level expression of ERBB2. Low ERBB2 expression is defined as ERBB2 immunohistochemical expression of 1+ or 2+ with a negative ERBB2 amplification by in situ hybridization. The efficacy of the antibody-drug conjugate trastuzumab deruxtecan in low-ERBB2, HR-positive mBC has been practice changing. However, there are conflicting data on the prognostic value of low ERBB2 expression in HR-positive mBC and whether low ERBB2 expression is a separate entity. To examine whether outcomes differ by immunohistochemical analysis for patients with HR-positive mBC with low ERBB2 expression vs those without ERBB2 expression when treated with targeted therapy (TT) plus endocrine therapy (ET). This single-institution cohort study used prospectively collected electronic data from the MD Anderson Cancer Center for patients with a diagnosis of HR-positive mBC treated with ET in combination with a TT (cyclin-dependent kinase 4/6 inhibitors [CDK4/6is], everolimus, or alpelisib) between January 1, 2010, and December 31, 2021. HR-positive mBC with either low or no ERBB2 expression. The main outcomes were median progression-free survival and overall survival. Data on demographic characteristics, estrogen and progesterone receptor status, ERBB2 status, histologic subtype, menopausal status, treatment duration, and survival status were collected. A total of 1585 women (median [range] age, 51 [24-92] years) were included in the study. Of these women, 1013 (63.9%) had mBC with low ERBB2 expression and 572 (36.1%) had mBC with no ERBB2 expression. A total of 1084 (68.4%) were treated with a CDK4/6i (912 patients were treated in the first line and 172 were treated in the second line); 475 (30.0%) received everolimus and 26 (1.6%) received alpelisib. In the patients who received a first-line CDK4/6i, 618 (67.8%) received an aromatase inhibitor as their ET backbone and 265 (29.1%) received fulvestrant. With a median follow-up time of 17.9 months (range, 1-111 months), progression-free survival and overall survival were not statistically different between the patients with low and no ERBB2 expression treated with TT plus ET. In this cohort study of patients with HR-positive mBC treated with TT plus ET, low ERBB2 expression did not have a significant association with prognosis.

FGFR2 mutations promote endometrial cancer progression through dual engagement of EGFR and Notch signalling pathways.

Mutations in the receptor tyrosine kinase gene fibroblast growth factor receptor 2 (FGFR2) occur at a high frequency in endometrial cancer (EC) and have been linked to advanced and recurrent disease. However, little is known about how these mutations drive carcinogenesis. Differential transcriptomic analysis and two-step quantitative real-time PCR (qRT-PCR) assays were applied to identify genes differentially expressed in two cohorts of EC patients carrying mutations in the FGFR2 gene as well as in EC cells harbouring mutations in the FGFR2. Candidate genes and target signalling pathways were investigated by qRT-PCR assays, immunohistochemistry and bioinformatics analysis. The functional roles of differently regulated genes were analysed using in vitro and in vivo experiments, including 3D-orthotypic co-culture systems, cell proliferation and migration protocols, as well as colony and focus formation assays together with murine xenograft tumour models. The molecular mechanisms were examined using CRISPR/Cas9-based loss-of-function and pharmacological approaches as well as luciferase reporter techniques, cell-based ectodomain shedding assays and bioinformatics analysis. We show that common FGFR2 mutations significantly enhance the sensitivity to FGF7-mediated activation of a disintegrin and metalloprotease (ADAM)17 and subsequent transactivation of the epidermal growth factor receptor (EGFR). We further show that FGFR2 mutants trigger the activation of ADAM10-mediated Notch signalling in an ADAM17-dependent manner, highlighting for the first time an intimate cooperation between EGFR and Notch pathways in EC. Differential transcriptomic analysis in EC cells in a cohort of patients carrying mutations in the FGFR2 gene identified a strong association between FGFR2 mutations and increased expression of members of the Notch pathway and ErbB receptor family. Notably, FGFR2 mutants are not constitutively active but require FGF7 stimulation to reprogram Notch and EGFR pathway components, resulting in ADAM17-dependent oncogenic growth. These findings highlight a pivotal role of ADAM17 in the pathogenesis of EC and provide a compelling rationale for targeting ADAM17 protease activity in FGFR2-driven cancers.

PO-02-214 JK07 PREVENTS HYPERTENSION-INDUCED ATRIAL FIBROSIS AND ATRIAL FIBRILLATION INDUCIBILITY IN A PIG MODEL

An important substrate of atrial fibrillation (AF) is atrial fibrosis, and a higher burden of fibrosis correlates with worse prognosis and therapy resistance. Currently, no therapies exist that target atrial fibrosis. JK07 is a long-acting neuregulin-fusion protein that activates the ERBB4 receptor, which mitigates ventricular fibrosis in models of heart failure. To test the ability of JK07 to reduce atrial fibrosis and AF inducibility in the minipig model of deoxycorticosterone acetate (DOCA, an aldosterone agonist) induced hypertension. Eighteen Aachener minipigs were randomized into three groups: control (CTRL), DOCA + Vehicle (DOCA+VEH) and DOCA+JK07. The control group did not undergo disease induction or therapeutic intervention. To induce hypertension and atrial fibrosis in minipigs in the DOCA+VEH and DOCA+JK07 groups, DOCA pellets were implanted releasing 10 mg/kg DOCA over a period of 60 days. The DOCA-implanted animals underwent weekly treatment with JK07 (0.3 mg/kg; DOCA + JK07) or vehicle control (DOCA + VEH), starting on the day of implantation (total of 9 administrations). After 60 days, arterial blood pressure was measured invasively and a decapolar catheter was placed in the right atrium. AF inducibility was tested by performing 50 burst pacing episodes and quantified as the percentage of “successful” episodes where an AF run ≥ 5 sec could be induced after the burst, out of the 50 attempts. Atrial fibrosis was quantified using AFAT software on Masson trichrome staining of left atrial appendage specimens, isolated after euthanizing the animals. Mean arterial pressure was significantly higher in the DOCA+VEH (142 ± 10 mmHg) and DOCA+JK07 (132 ± 15 mmHg) groups than in the CTRL group (105 ± 8 mmHg, p<0.01), without an effect of JK07. AF inducibility in the DOCA+VEH group was significantly higher than in the CTRL group (66/250 vs. 9/300, p<0.001), and the DOCA + JK07 group (66/250 vs. 8/300, p<0.001). Likewise, the degree of atrial fibrosis was significantly higher in the DOCA+VEH group compared to CTRL (10.32% ± 4.74% surface area vs. 5.28% ± 3.06%, p<0.001) and compared to DOCA+JK07 (10.32% ± 4.74% vs. 3.82% ± 2.68%, p<0.001). JK07 prevents atrial fibrosis and AF inducibility in a porcine DOCA model. The effect of JK07 is unrelated to effects on blood pressure, but probably related to reduced atrial fibrogenesis.

In vivo tissue pharmacokinetics of ERBB2-specific binding oligonucleotide based drugs by PET imaging.

Although aptamers have shown excellent target specificity in preclinical and clinical studies either by themselves or as aptamer-drug conjugates, their in vivo tissue pharmacokinetic (PK) analysis is still problematic. We aimed to examine the utility of image based positron emission tomography (PET) to evaluate in vivo tissue PK, target specificity, and applicability of oligonucleotides. For this, fluorine-18-labeled aptamers with erb-b2 receptor tyrosine kinase 2 (ERBB2)-specific binding were synthesized by base-pair hybridization using a complementary oligonucleotide platform. To investigate the PKs and properties in vivo tissue, usefulness of in vivo PET imaging in the development of oligonucleotide based drug as an assessment tool was evaluated in normal and tumor xenografted mice. ERBB2-cODN-idT-APs-[18 F]F ([18 F]1), injected intravenously showed significant and rapid uptake in most tissues except for the initial brain and muscle; the uptake was highest in the heart, followed by kidneys, liver, lungs, gall bladder, spleen, and stomach. The main route of excretion was through the renal approximately 77.8% while about 8.3% through the biliary tract of the total dose. The estimated effective dose for an adult woman was 0.00189 mGy/MBq, which might be safe. ERBB2-positive tumor could be well visualized in KPL4 xenograft animal model by in vivo PET imaging. Consequently, the distribution in each organ including ERBB2 expression could be well determined and quantified by PET with fluorine-18-labeled aptamers. In vivo PK parameters such as T1/2 , Tmax , AUC, and Cmax , were also successfully estimated. These results suggest that image based PET with radioisotope-labeled aptamers could be provide valuable information on properties of oligonucleotide based drugs in drug discovery of targeted therapeutics against various diseases.

Developmental loss of ErbB4 in PV interneurons disrupts state-dependent cortical circuit dynamics.

GABAergic inhibition plays an important role in the establishment and maintenance of cortical circuits during development. Neuregulin 1 (Nrg1) and its interneuron-specific receptor ErbB4 are key elements of a signaling pathway critical for the maturation and proper synaptic connectivity of interneurons. Using conditional deletions of the ERBB4 gene in mice, we tested the role of this signaling pathway at two developmental timepoints in parvalbumin-expressing (PV) interneurons, the largest subpopulation of cortical GABAergic cells. Loss of ErbB4 in PV interneurons during embryonic, but not late postnatal development leads to alterations in the activity of excitatory and inhibitory cortical neurons, along with severe disruption of cortical temporal organization. These impairments emerge by the end of the second postnatal week, prior to the complete maturation of the PV interneurons themselves. Early loss of ErbB4 in PV interneurons also results in profound dysregulation of excitatory pyramidal neuron dendritic architecture and a redistribution of spine density at the apical dendritic tuft. In association with these deficits, excitatory cortical neurons exhibit normal tuning for sensory inputs, but a loss of state-dependent modulation of the gain of sensory responses. Together these data support a key role for early developmental Nrg1/ErbB4 signaling in PV interneurons as a powerful mechanism underlying the maturation of both the inhibitory and excitatory components of cortical circuits.

Pattern of MUC6 expression across 119 different tumor types: A tissue microarray study on 15 412 tumors.

Mucin 6 (MUC6) is a secreted gel-forming mucin covering the surfaces of gastrointestinal and other tissues. Published work demonstrates that MUC6 can also be expressed in several cancer types and can aid in the distinction of different tumor entities. To systematically analyze MUC6 expression in normal and cancerous tissues, a tissue microarray containing 15 412 samples from 119 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. At least a weak MUC6 positivity was seen in 50 of 119 (42%) tumor entities. Thirty-three tumor entities included tumors with strong positivity. MUC6 immunostaining was most frequent in mucinous carcinomas of the breast (44%), adenocarcinomas of the stomach (30%-40%) and esophagus (35%), and neuroendocrine carcinomas of the colon. Strong MUC6 staining was linked to advanced pT stage (p = 0.0464), defective mismatch repair status and right-sided tumor location (p < 0.0001 each) in colorectal cancer, as well as to high tumor grade (p = 0.0291), nodal metastasis (p = 0.0485), erb-b2 receptor tyrosine kinase 2 positivity (p < 0.0001) and negative estrogen receptor (p = 0.0332)/progesterone receptor (p = 0.0257) status in breast carcinomas of no special type. The broad range of tumor types with MUC6 expression limits the utility of MUC6 immunohistochemistry for the distinction of different tumor types.

Feedback activation of EGFR/wild-type RAS signaling axis limits KRASG12D inhibitor efficacy in KRASG12D-mutated colorectal cancer

Colorectal cancer (CRC), which shows a high degree of heterogeneity, is the third most deadly cancer worldwide. Mutational activation of KRASG12D occurs in approximately 10–12% of CRC cases, but the susceptibility of KRASG12D-mutated CRC to the recently discovered KRASG12D inhibitor MRTX1133 has not been fully defined. Here, we report that MRTX1133 treatment caused reversible growth arrest in KRASG12D-mutated CRC cells, accompanied by partial reactivation of RAS effector signaling. Through a drug-anchored synthetic lethality screen, we discovered that epidermal growth factor receptor (EGFR) inhibition was synthetic lethal with MRTX1133. Mechanistically, MRTX1133 treatment downregulated the expression of ERBB receptor feedback inhibitor 1 (ERRFI1), a crucial negative regulator of EGFR, thereby causing EGFR feedback activation. Notably, wild-type isoforms of RAS, including H-RAS and N-RAS, but not oncogenic K-RAS, mediated signaling downstream of activated EGFR, leading to RAS effector signaling rebound and reduced MRTX1133 efficacy. Blockade of activated EGFR with clinically used antibodies or kinase inhibitors suppressed the EGFR/wild-type RAS signaling axis, sensitized MRTX1133 monotherapy, and caused the regression of KRASG12D-mutant CRC organoids and cell line-derived xenografts. Overall, this study uncovers feedback activation of EGFR as a prominent molecular event that restricts KRASG12D inhibitor efficacy and establishes a potential combination therapy consisting of KRASG12D and EGFR inhibitors for patients with KRASG12D-mutated CRC.

Proteolysis Targeting Chimera (PROTAC) as a promising novel therapeutic modality for the treatment of triple-negative breast cancer (TNBC).

Breast cancer (BC) is considered one of the most prevalent malignancies impacting women worldwide, constituting 15% of all new cancer cases. It is classified according to its molecular targets into three subtypes; HR+/ERBB2-, ERBB2+/HR+, or HR- and triple-negative breast cancer (TNBC). TNBC is considered aggressive cancer with bad prognosis and poor clinical outcomes. It lacks estrogen, progesterone, and ERBB2 receptors rendering its treatment more challenging. Owing to its unresponsiveness to hormonal therapy, quick tumor growth, and the high probability to have spread at the time of discovery, it is more likely to recur following therapy. Proteolysis Targeting Chimeric molecules (PROTACs) are hetero-bifunctional molecules that are able to hijack the ubiquitin-proteasome system, a major physiological proteolytic mechanism. This leads to ubiquitination through endogenous E3 ligases and subsequent degradation through 26-S proteasome. Since its discovery in 2001, PROTACs have been considered an important therapeutic modality due to their ability to target and degrade undruggable proteins. In vitro studies have been conducted to investigate the possibility of using PROTACs as a therapeutic modality in TNBC. Data available propose great potential of PROTACs technology as a major candidate in TNBC management. Further in vitro and in vivo clinical trials are required to establish a definitive decision. In this review, we aim to give a brief overview of TNBC and PROTACs and highlight the rationale behind using PROTACs as a therapeutic modality in patients with TNBC.

Abstract 2565: Divergentnucleocytoplasmic transport influences escape from HER2-activated DCIS-like state

Abstract HER2/ErbB2 and EGFR/ErbB1 activation incite a phenotype resembling premalignant escape from ductal carcinoma in situ (DCIS) in the 3D multicellular arrangement of breast-epithelial cells. This phenomenon tends to be infrequent, and the exact mechanism for its heterogeneous presentation has been elusive. We sought to investigate the process by which ErbB receptors facilitate a picture of incomplete penetrance in DCIS escape (DE). To identify the transcriptional signatures that prime cells towards DE, we randomly profiled 10-cell transcriptomes of single spheroids after 24 hours of ERBB activation, and frequency matched gene expression to the DE-phenotype. We uncovered a network of nucleocytoplasmic transport (NCT) regulators that alter the DE-phenotype penetrance. Of the regulators, CSE1L (an exportin) synergistically increases the DE-frequency when induced, while its knockdown reduces the phenotype, suggesting its functional role. Furthermore, we evaluated the significance of this result in vivo by perturbing Cse1l in Erbb2-amplified mouse mammary-gland tumors and found that activation of Erbb2 (mimicked by ERBB-inhibitor release) alters the macroscale organization of tumors, reminiscent of DE. Specifically, we found an elevated CSE1L inhibits an accumulation of classical nuclear-localizing cargoes that require binding with importin-α/β (KPNAs, KPNB1) complexes. Using proximity labeling with BirA*-fused with CSE1L, we identified ERBB1/ERBB2/ERBB4 as CSE1L interactors. Mechanistically, hetero-dimerized ERBB receptors interact with importin-α/β complexes and re-localize to the nucleus, corroborating previous observations of receptors’ nuclear translocation. Intriguingly, we observed that Doxycycline-induced knockdown of clathrin heavy chain (CLTC, involved in intracellular trafficking of receptors via endocytosis) synergizes with ERBB activation to increase the DE-phenotype penetrance. Using ChIP-Seq, we found that ERBB1 binds to the locus of microRNA, miR205, which negatively regulates the expression of importin-α, mainly KPNA1. These results indicate that ERBB receptors translocated to the nucleus reduce the DE-phenotype by inhibiting importins via miR205, while CSE1L induction relieves this inhibition by reducing the receptor's nuclear accumulation. Taken together, these results show that nucleocytoplasmic shuttling of receptors in ERBB-active breast epithelia generates tunable cross-inhibitory feedback through nucleocytoplasmic transport that results in long-term, heterogeneous multicellular fates. Citation Format: Lixin Wang, Bishal Paudel, Anthony McKnight, Kevin Janes. Divergentnucleocytoplasmic transport influences escape from HER2-activated DCIS-like state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2565.