ErbB Inhibitors Research Articles

Coordinate transcriptional regulation of ErbB2/3 by C-terminal binding protein 2 signals sensitivity to ErbB2 inhibition in pancreatic adenocarcinoma

There is a critical need to identify new therapeutic vulnerabilities in pancreatic ductal adenocarcinoma (PDAC). Transcriptional co-regulators C-terminal binding proteins (CtBP) 1 and 2 are highly overexpressed in human PDAC, and CRISPR-based homozygous deletion of Ctbp2 in a mouse PDAC cell line (CKP) dramatically decreased tumor growth, reduced metastasis, and prolonged survival in orthotopic mouse allografts. Transcriptomic profiling of tumors derived from CKP vs. Ctbp2-deleted CKP cells (CKP/KO) revealed significant downregulation of the EGFR-superfamily receptor Erbb3, the heterodimeric signaling partner for both EGFR and ErbB2. Compared with CKP cells, CKP/KO cells also demonstrated reduced Erbb2 expression and did not activate downstream Akt signaling after stimulation of Erbb3 by its ligand neuregulin-1. ErbB3 expression in human PDAC cell lines was similarly dependent on CtBP2 and depletion of ErbB3 in a human PDAC cell line severely attenuated growth, demonstrating the critical role of ErbB3 signaling in maintaining PDAC cell growth. Sensitivity to the ErbB2-targeted tyrosine kinase inhibitor lapatinib, but not the EGFR-targeted agent erlotinib, varied in proportion to the level of ErbB3 expression in mouse and human PDAC cells, suggesting that an ErBb2 inhibitor can effectively leverage CtBP2-driven transcriptional activation of physiologic ErbB2/3 expression and signaling in PDAC cells for therapeutic benefit.

ERBB4-Mediated Signaling Is a Mediator of Resistance to PI3K and BTK Inhibitors in B-cell Lymphoid Neoplasms.

BTK and PI3K inhibitors are among the drugs approved for the treatment of patients with lymphoid neoplasms. Although active, their ability to lead to long-lasting complete remission is rather limited, especially in the lymphoma setting. This indicates that tumor cells often develop resistance to the drugs. We started from a marginal zone lymphoma cell line, Karpas-1718, kept under prolonged exposure to the PI3Kδ inhibitor idelalisib until acquisition of resistance, or with no drug. Cells underwent transcriptome, miRNA and methylation profiling, whole-exome sequencing, and pharmacologic screening, which led to the identification of the overexpression of ERBB4 and its ligands HBEGF and NRG2 in the resistant cells. Cellular and genetic experiments demonstrated the involvement of this axis in blocking the antitumor activity of various BTK/PI3K inhibitors, currently used in the clinical setting. Addition of recombinant HBEGF induced resistance to BTK/PI3K inhibitors in parental cells and in additional lymphoma models. Combination with the ERBB inhibitor lapatinib was beneficial in resistant cells and in other lymphoma models already expressing the identified resistance factors. An epigenetic reprogramming sustained the expression of the resistance-related factors, and pretreatment with demethylating agents or EZH2 inhibitors overcame the resistance. Resistance factors were also shown to be expressed in clinical specimens. In conclusion, we showed that the overexpression of ERBB4 and its ligands represents a novel mechanism of resistance for lymphoma cells to bypass the antitumor activity of BTK and PI3K inhibitors and that targeted pharmacologic interventions can restore sensitivity to the small molecules.

Radiotherapy Plus Cisplatin With or Without Lapatinib for Non–Human Papillomavirus Head and Neck Carcinoma

Patients with locally advanced non-human papillomavirus (HPV) head and neck cancer (HNC) carry an unfavorable prognosis. Chemoradiotherapy (CRT) with cisplatin or anti-epidermal growth factor receptor (EGFR) antibody improves overall survival (OS) of patients with stage III to IV HNC, and preclinical data suggest that a small-molecule tyrosine kinase inhibitor dual EGFR and ERBB2 (formerly HER2 or HER2/neu) inhibitor may be more effective than anti-EGFR antibody therapy in HNC. To examine whether adding lapatinib, a dual EGFR and HER2 inhibitor, to radiation plus cisplatin for frontline therapy of stage III to IV non-HPV HNC improves progression-free survival (PFS). This multicenter, phase 2, double-blind, placebo-controlled randomized clinical trial enrolled 142 patients with stage III to IV carcinoma of the oropharynx (p16 negative), larynx, and hypopharynx with a Zubrod performance status of 0 to 1 who met predefined blood chemistry criteria from October 18, 2012, to April 18, 2017 (median follow-up, 4.1 years). Data analysis was performed from December 1, 2020, to December 4, 2020. Patients were randomized (1:1) to 70 Gy (6 weeks) plus 2 cycles of cisplatin (every 3 weeks) plus either 1500 mg per day of lapatinib (CRT plus lapatinib) or placebo (CRT plus placebo). The primary end point was PFS, with 69 events required. Progression-free survival rates between arms for all randomized patients were compared by 1-sided log-rank test. Secondary end points included OS. Of the 142 patients enrolled, 127 (median [IQR] age, 58 [53-63] years; 98 [77.2%] male) were randomized; 63 to CRT plus lapatinib and 64 to CRT plus placebo. Final analysis did not suggest improvement in PFS (hazard ratio, 0.91; 95% CI, 0.56-1.46; P = .34) or OS (hazard ratio, 1.06; 95% CI, 0.61-1.86; P = .58) with the addition of lapatinib. There were no significant differences in grade 3 to 4 acute adverse event rates (83.3% [95% CI, 73.9%-92.8%] with CRT plus lapatinib vs 79.7% [95% CI, 69.4%-89.9%] with CRT plus placebo; P = .64) or late adverse event rates (44.4% [95% CI, 30.2%-57.8%] with CRT plus lapatinib vs 40.8% [95% CI, 27.1%-54.6%] with CRT plus placebo; P = .84). In this randomized clinical trial, dual EGFR-ERBB2 inhibition with lapatinib did not appear to enhance the benefit of CRT. Although the results of this trial indicate that accrual to a non-HPV HNC-specific trial is feasible, new strategies must be investigated to improve the outcome for this population with a poor prognosis. ClinicalTrials.gov Identifier: NCT01711658.

Targeting collagen XVIII improves the efficiency of ErbB inhibitors in breast cancer models.

The tumor extracellular matrix (ECM) critically regulates cancer progression and treatment response. Expression of the basement membrane component collagen XVIII (ColXVIII) is induced in solid tumors, but its involvement in tumorigenesis has remained elusive. We show here that ColXVIII was markedly upregulated in human breast cancer (BC) and was closely associated with a poor prognosis in high-grade BCs. We discovered a role for ColXVIII as a modulator of epidermal growth factor receptor tyrosine kinase (ErbB) signaling and show that it forms a complex with ErbB1 and -2 (also known as EGFR and human epidermal growth factor receptor 2 [HER2]) and α6-integrin to promote cancer cell proliferation in a pathway involving its N-terminal portion and the MAPK/ERK1/2 and PI3K/AKT cascades. Studies using Col18a1 mouse models crossed with the mouse mammary tumor virus-polyoma virus middle T antigen (MMTV-PyMT) mammary carcinogenesis model showed that ColXVIII promoted BC growth and metastasis in a tumor cell-autonomous manner. Moreover, the number of mammary cancer stem cells was significantly reduced in the MMTV-PyMT and human cell models upon ColXVIII inhibition. Finally, ablation of ColXVIII substantially improved the efficacy of ErbB-targeting therapies in both preclinical models. In summary, ColXVIII was found to sustain the stemness properties of BC cells and tumor progression and metastasis through ErbB signaling, suggesting that targeting ColXVIII in the tumor milieu may have important therapeutic potential.

Histology-agnostic tumor treatment - a farewell to tumor entities?

Considerable efforts concerning the molecular characterization and targeted treatment of cancer have significantly improved treatment options and prognosis of tumor patients. Nevertheless, in tumor entities without recurrent genetic alterations the application of molecular testing for potentially targetable lesions remains heterogeneous and, in most cases, the approval of targeted therapies is still restricted to defined tumor entities harboring corresponding predictive biomarkers.The broad genomic analysis of different tumor entities including rare cancers within several genome sequencing initiatives and precision oncology programs has revealed the occurrence of addressable molecular alterations across many tumor entities, although their incidence may differ significantly in the context of the underlying cancer type. The treatment of molecularly defined patient cohorts demonstrated an impressive tumor-agnostic efficacy of certain therapeutics such as NTRK inhibitors, while the outcome of other targeted therapies, such as ERBB or BRAF inhibitors, varied in the context of the underlying disease.In the meantime, a handful targeted therapeutics addressing NRTK and RET fusions, the BRAF V600E mutation or different features of defective DNA mismatch repair and high tumor mutational burden has been approved for histology-agnostic treatment of tumors harboring these target lesions. Ongoing molecularly stratified basket trials will further investigate the tumor-agnostic efficacy of different targeted treatment approaches.

Single cell profiling of female breast fibroadenoma reveals distinct epithelial cell compositions and therapeutic targets

Fibroadenomas (FAs) are the most common breast tumors in women. No pharmacological agents are currently approved for FA intervention owing to its unclear mechanisms and a shortage of reproducible human models. Here, using single-cell RNA sequencing of human FAs and normal breast tissues, we observe distinct cellular composition and epithelial structural changes in FAs. Interestingly, epithelial cells exhibit hormone-responsive functional signatures and synchronous activation of estrogen-sensitive and hormone-resistant mechanisms (ERBB2, BCL2 and CCND1 pathways). We develop a human expandable FA organoid system and observe that most organoids seem to be resistant to tamoxifen. Individualized combinations of tamoxifen with ERBB2, BCL2 or CCND1 inhibitors could significantly suppress the viability of tamoxifen-resistant organoids. Thus, our study presents an overview of human FA at single-cell resolution that outlines the structural and functional differences between FA and normal breast epithelium and, in particular, provides a potential therapeutic strategy for breast FAs.

Inhibition of Erb-B2 Receptor Tyrosine Kinase 3 and Associated Regulatory Pathways Potently Impairs Malignant Peripheral Nerve Sheath Tumor Proliferation and Survival

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, currently untreatable Schwann cell-derived neoplasms with hyperactive mitogen-activated protein kinase and mammalian target of rapamycin signaling pathways. To identify potential therapeutic targets, previous studies used genome-scale shRNA screens that implicated the neuregulin-1 receptor erb-B2 receptor tyrosine kinase 3 (erbB3) in MPNST proliferation and/or survival. The current study shows that erbB3 is commonly expressed in MPNSTs and MPNST cell lines and that erbB3 knockdown inhibits MPNST proliferation and survival. Kinomic and microarray analyses of Schwann and MPNST cells implicate Src- and erbB3-mediated calmodulin-regulated signaling as key pathways. Consistent with this, inhibition of upstream (canertinib, sapitinib, saracatinib, and calmodulin) and parallel (AZD1208) signaling pathways involving mitogen-activated protein kinase and mammalian target of rapamycin reduced MPNST proliferation and survival. ErbB inhibitors (canertinib and sapitinib) or erbB3 knockdown in combination with Src (saracatinib), calmodulin [trifluoperazine (TFP)], or proviral integration site of Moloney murine leukemia kinase (AZD1208) inhibition even more effectively reduces proliferation and survival. Drug inhibition enhances an unstudied calmodulin-dependent protein kinase IIα phosphorylation site in an Src-dependent manner. The Src family kinase inhibitor saracatinib reduces both basal and TFP-induced erbB3 and calmodulin-dependent protein kinase IIα phosphorylation. Src inhibition (saracatinib), like erbB3 knockdown, prevents these phosphorylation events; and when combined with TFP, it even more effectively reduces proliferation and survival compared with monotherapy. These findings implicate erbB3, calmodulin, proviral integration site of Moloney murine leukemia kinases, and Src family members as important therapeutic targets in MPNSTs and demonstrate that combinatorial therapies targeting critical MPNST signaling pathways are more effective.

Recombinant human neuregulin-1 alleviates immobilization-induced neuromuscular dysfunction via neuregulin-1/ErbB signaling pathway in rat

Immobilization-induced Neuromuscular Dysfunction (NMD) increases morbidity and mortality of patients in Intensive Care Units. However, the underlying mechanism of NMD remain poorly elucidated which limited the development of therapeutic method for NMD. Here we developed an immobilization rat model and tested the hypothesis that decreased expression of NRG-1, abnormal expression and distribution of nicotinic acetylcholine receptors (nAChRs) in skeletal muscle caused by immobilization can lead to NMD. To investigate the role of NRG-1/ErbB pathway on immobilization-induced NMD, exogenous recombinant human neuregulin-1 (rhNRG-1) was used to increase the expression of NRG-1 in skeletal muscle during immobilization. It was observed rhNRG-1 significantly alleviated the muscle loss and enhanced the expression of ε-nAChR, while diminished the expression of γ- and α7-nAChR and NMD. Interestingly, ErbB inhibitor PD158780 blocked the protective effects of rhNRG-1. Collectively, the results of present study suggested that rhNRG-1 attenuated immobilization-induced muscle loss and NMD, suppressed γ- and α7-nAChR production, enhanced ε-nAChR synthesis via activating NRG-1/ErbB pathway. Taken together, our findings provide novel insights into NMD contribution, suggesting that the rhNRG-1 is a promising therapy to protect against immobilization-induced myopathy.

Afatinib combined with anti-PD1 enhances immunotherapy of hepatocellular carcinoma via ERBB2/STAT3/PD-L1 signaling.

Afatinib is mainly used to treat advanced non-small cell lung cancer, but its therapeutic effect on hepatocellular carcinoma is still unclear. Over 800 drugs were screened by CCK8 technology and afatinib was found to have a significant inhibitory effect on liver cancer cells. The expression of PDL1 in tumor cells treated with drugs were detected by qRT-PCR and Weston Blot experiments. The effects of afatinib on the growth, migration and invasion of HCC cells were evaluated using wound healing, Transwell, and cell cloning assays. The in vivo effects of afatinib in combination with anti-PD1 were evaluated in C57/BL6J mice with subcutaneous tumorigenesis. Bioinformatics analysis was performed to explore the specific mechanism of afatinib's inhibition of ERBB2 in improving the expression level of PD-L1, which was subsequently verified through experiments. Afatinib was found to have a significant inhibitory effect on liver cancer cells, as confirmed by in vitro experiments, which demonstrated that it could significantly suppress the growth, invasion and migration of HCC cells. qRT PCR and Weston Blot experiments also showed that Afatinib can enhance the expression of PD-L1 in tumor cells. In addition, in vitro experiments confirmed that afatinib can significantly enhance the immunotherapeutic effect of hepatocellular carcinoma. Afatinib's ability to increase PD-L1 expression is mediated by STAT3 activation following its action on HCC cells. Afatinib enhances PD-L1 expression in tumor cells through the STAT3/PD-L1 pathway. The combination of afatinib and anti-PD1 treatment significantly increases the immunotherapeutic effect of HCC.

Abstract 394: ERBB4-mediated signaling is a mediator of resistance to BTK and PI3K inhibitors in B cell lymphoid neoplasms

Abstract Background: Marginal zone lymphoma (MZL) is an indolent yet incurable B cell malignancy. Two BTK inhibitors, ibrutinib and zanubrutinib, are FDA approved for relapsed/refractory MZL patients. PI3K inhibitors have also shown clinical activity. The identification of the mechanisms of resistance can provide useful information to optimize the use of the agents. We previously reported an IL6 driven MZL model of PI3K inhibitors resistance developed by prolonged exposure to the PI3Kδ inhibitor idelalisib (Arribas, Haematologica 2022). Here, we present the detailed characterization of a second model with resistance to both BTK and PI3K inhibitors. Methods: MTT assay. RNA-Seq, whole exome sequencing, miRNA and methylation profiling. FACS and ELISA analyses. Results: Resistant cells, developed by continuous exposure of the cell line Karpas1718 to idelalisib, showed resistance to various inhibitors of BTK (ibrutinib, zanubrutinib, acalabrutinib and pirtobrutinib) and PI3K (idelalisib, duvelisib, copanlisib and umbralisib). No mutations affecting BTK, PLCG2 or CXCR4 were identified in resistant cells, which had higher expression of genes involved in ERBB signaling (HBEGF, NRG2, ERBB4), cell proliferation (PBK, MKI67, TCL1A) and DNA recombination (RAG1, RAG2) than parental cells. We confirmed cell surface ERBB4 up-regulation, and the cytoplasmatic expression and secretion of its ligand HBEGF in resistant cells, which led to increased levels of p-AKT and p-ERK. The miRNAs miR-29c and let-7c, known negative regulators of the HBEGF-ERBB axis, were fully methylated and down-regulated in resistant compared to parental cells. ERBB4 genetic silencing improved sensitivity to PI3Kδ inhibitor, and exposure to let-7c or miR-29c mimics decreased secreted HBEGF and recovered sensitivity to PI3K inhibitors in resistant cells. Addition of recombinant HBEGF (rHBEGF) induced resistance to BTK and to PI3K inhibitors in parental cells and in other lymphoma models including mantle cell lymphomas and diffuse large B cell lymphomas (DLBCL). The rHBEGF induced resistance was reverted adding the ERBB inhibitor lapatinib. To extend our findings to the clinical context, using two MZL and one DLBCL expression datasets, we showed HBEGF and ERBB4 expression in clinical specimens. Finally, HBEGF levels appeared elevated in the serum of CLL patients with primary or acquired resistance to PI3Kδ or to BTK inhibitors, compared to patients responding to the drugs and paired for similar clinical features. Conclusions: We characterized a novel B cell lymphoma model of secondary resistance to BTK and PI3K inhibitors. Our results indicate that epigenetic plasticity led to the activation of HBEGF-ERBB signaling sustaining resistance to BTK/PI3K inhibitors, which can be overcome using epigenetic agents and ERBB inhibitors. These therapeutics approaches could be tested in novel clinical trials. AJA, SN: equally contributed. Citation Format: Alberto J. Arribas, Sara Napoli, Luciano Cascione, Laura Barnabei, Giulio Sartori, Eleonora Cannas, Eugenio Gaudio, Chiara Tarantelli, Afua A. Mensah, Filippo Spriano, Antonella Zucchetto, Francesca M. Rossi, Andrea Rinaldi, Manuel Castro de Moura, Anastasios Stathis, Georg Stussi, Valter Gattei, Jennifer R. Brown, Manel Esteller, Emanuele Zucca, Davide Rossi, Francesco Bertoni. ERBB4-mediated signaling is a mediator of resistance to BTK and PI3K inhibitors in B cell lymphoid neoplasms [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 394.

Abstract 4008: TAS2940, a novel brain-penetrable pan-ERBB inhibitor, exhibits tumor regression and prolongs survival in intracranial models bearing ERBB aberrations

Abstract Background: Primary and metastatic brain tumors have significant unmet medical needs as few drugs can penetrate the blood-brain barrier (BBB). Brain metastasis is more common in non-small cell lung cancer (NSCLC) and breast cancer than in other cancers. Genetic alterations in ERBB family genes are frequently observed in those cancers, with about half of HER2/EGFR aberrations metastasizing in the brain. EGFR aberrations have been also observed in glioblastoma. Several pan-ERBB inhibitors are clinically available or under clinical development, such as afatinib, neratinib, and poziotinib. However, none are highly brain-penetrable as they are pumped out via the BBB. Here, we report TAS2940 as a novel potent pan-ERBB inhibitor that elicits brain penetrability and anti-tumor effects in brain metastasis models. Methods: The kinase selectivity of TAS2940 was evaluated in a panel of 257 kinases. Autophosphorylation of wild-type and mutant ERBB in stably expressing cell lines was evaluated using a quantitative immunofluorescence assay. The growth inhibitory effect on cell lines expressing HER2/EGFR aberrations was evaluated based on quantitation of the cellular ATP concentration. Cell lines were transplanted subcutaneously or intracranially in BALB/cAJcl-nu/nu mice. HER2 tyrosine kinase inhibitors, including TAS2940, were then orally administered once daily followed by evaluation of tumor volume and body weight. Results: TAS2940 showed potent and selective inhibition against ERBB family proteins over more than 250 kinases on enzymatic assay. TAS2940 also showed preferential cytotoxicity in cells harboring HER2/EGFR genetic alterations in vitro, while sparing cells without ERBB genetic aberrations. TAS2940 showed potent anti-proliferative activity in cells containing HER2 genetic alterations or EGFR exon 20 insertions, commonly found in NSCLC or breast cancer, as well as EGFRvIII alterations, frequently observed in glioblastoma. In vivo xenograft tumor models with various EGFR genetic alterations showed that TAS2940 exerted anti-tumor effects, such as growth retardation or regression. We further examined brain penetration by assessing the brain to blood concentration of TAS2940 in mice with intact BBBs, showing higher brain penetrability compared to other ERBB inhibitors. Evaluation of TAS2940 in intracranial brain metastasis mouse models resulted in tumor regression and prolonged survival. Conclusion: TAS2940 is a novel potent and selective pan-ERBB inhibitor with high brain penetrability in vivo. These results support the clinical development of TAS2940 in primary and metastatic brain tumors driven by ERBB genetic aberrations. Citation Format: Kei Oguchi, Hikari Araki, Shingo Tsuji, Masayuki Nakamura, Akihiro Miura, Kaoru Funabashi, Akiko Osada, Sakiho Tanaka, Takamasa Suzuki, Shinji Mizuarai. TAS2940, a novel brain-penetrable pan-ERBB inhibitor, exhibits tumor regression and prolongs survival in intracranial models bearing ERBB aberrations. [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 4008.

Abstract 3969: Screening of kinase inhibitors as bioenergetic metabolism modulator using the XF Real-Time ATP Rate Assay

Abstract Cancer cells are metabolically reprogrammed through tumor initiation and progression, and there are increasing developments in cancer therapies exploiting the potential metabolic vulnerability. A better understanding of cancer cell metabolic regulation can also contribute to modulating the harsh tumor microenvironment, which strongly affects the efficacy of emerging anti-cancer immunotherapies. As one of the critical contributors to cancer therapy design, there is increasing demand for time-efficient and reliable analytic tools when searching for drugs and genes modulating cancer cell metabolism. The XF Real-Time ATP Rate Assay measures ATP production from glycolysis and mitochondrial respiration simultaneously and provides a quantitative overview of the bioenergetic phenotype of the cells. By using ATP production rate as a universal unit of bioenergetic metabolism, this assay quickly detects metabolic modulations induced by chemical stimuli or genetic modifications. It allows the identification of potential targets for therapy design. In this study, we used the Seahorse XF Pro Analyzer combined with the XF ATP Rate assay to screen 80 kinase inhibitors for the suppressive effects on energy metabolism and/or inducing a metabolic phenotype shift in THP-1 cells, a monocytic leukemia cell line, using PBMC as the control. We identified four mitochondrial and two glycolytic suppressors using the screening and dose-response views available in Seahorse Analytics, a cloud-based data analysis tool for Seahorse XF assays. These compounds showed moderate or no effects on the net ATP production rates but induced a significant shift in the metabolic phenotype of THP-1 cells. Among the selected kinase inhibitors, AG-879 (ErbB2 inhibitor), SU-1498 (VEGFR inhibitor), and U-0126 (MEK1/2 inhibitor) showed the most potent inhibitory effect on mitochondrial energy production in THP-1 cells. Dose-response studies indicated that SU-1498 and U-0126 have higher potency in THP-1 than PBMC, and thus they can be considered good cancer-cell targets for mitochondrial suppressors. In contrast, AG-879 showed a similar or higher inhibitory effect on PBMC even though it is the most potent mitochondrial suppressor among the selected compounds. These results demonstrate that the XF Real-Time ATP Rate Assay can be used as a primary assay for screening and validating drug candidate(s) targeting the bioenergetic metabolism of cancer cells. Citation Format: Yoonseok Kam, Lisa Winer, Natalia Romero. Screening of kinase inhibitors as bioenergetic metabolism modulator using the XF Real-Time ATP Rate Assay. [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 3969.

Abstract 1440: Identification of a novel EGFR sparing brain penetrant ErbB2 inhibitor with activity against oncogenic ErbB2 mutations

Abstract Oncogenic driver mutations in ErbB2 (e.g., S310F/Y, L755S, V777L, V842I) are present in a variety of tumor types including gastric, uterine, urothelial carcinoma, non-small cell lung, breast, and colorectal, some of which are also known to metastasize to the CNS. These mutations can also emerge as mechanisms of acquired resistance to targeted therapies and as such represent a novel and actionable target mutation set across solid tumor indications. Current therapies that target ErbB2 have poor potency against these mutations or are limited in their utility due to inhibition of wild type epidermal growth factor receptors (WT EGFR). The inhibition of WT EGFR is associated with debilitating clinical effects that include diarrhea, stomatitis, and hepatotoxicity. Additionally, treatment of CNS metastases is especially challenging since the potential therapy must also cross the blood brain barrier to have a therapeutic effect. Next generation ErbB2 inhibitors that are active against ErbB2 mutations, spare WT EGFR, and that can penetrate the brain would provide improved target coverage with the potential for fewer off target side effects compared to current therapies and as well as the potential to exhibit superior clinical efficacy. Herein, we describe the pre-clinical in vitro and in vivo activity of a novel, brain penetrant ErbB2 inhibitor which retains potency against prevalent mutations while sparing WT EGFR. Citation Format: Mark J Chicarelli, Karyn Bouhana, Richard K. Brizendine, LouAnn Cable, Michelle Crow, Brad Fell, John Fischer, Jennifer Fulton, Anna Guarnieri, Ravi Jalluri, Hailey Knox, Keith A. Koch, Rob Rieger, John Robinson, Lee M. Stunkard, Francis Sullivan, Roy Turton, Shannon Winski, Yeyun Zhou. Identification of a novel EGFR sparing brain penetrant ErbB2 inhibitor with activity against oncogenic ErbB2 mutations [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 1440.

Metformin alleviates ethanol-induced cardiomyocyte injury by activating AKT/Nrf2 signaling in an ErbB2-dependent manner.

Metformin, a first-line oral anti-diabetic drug, has recently been reported to exert protective effect on various cardiovascular diseases. However, the potential role of metformin in ethanol-induced cardiomyocyte injury is still unknown. Therefore, this study was aimed to investigate the effect of metformin on ethanol-induced cardiomyocyte injury and its underlying mechanism. H9c2 cardiomyocytes were exposed to ethanol for 24h to establish an ethanol-induced cardiomyocyte injury model, and followed by treatment with metformin in the presence or absence of Lapatinib (an ErbB2 inhibition). CCK8 and LDH assays demonstrated that metformin improved cell viability in cardiomyocytes exposed to ethanol. Furthermore, metformin suppressed cardiomyocyte apoptosis and reduced the expressions of apoptosis-related proteins (Bax and C-CAS-3). In addition, our results showed that metformin activated the AKT/Nrf2 pathway, and then promoted Nrf2 nuclear translocation and the transcription of its downstream antioxidant genes (HO-1, CAT and SOD2), thereby inhibiting oxidative stress. Interestingly, we found that ErbB2 protein expression was significantly inhibited in ethanol-treated cardiomyocytes, which was markedly reversed by metformin. In contrast, Lapatinib largely abrogated the activation of AKT/Nrf2 signaling by metformin, accompanied by the increases in oxidative stress and cardiomyocyte apoptosis, indicating that metformin prevented ethanol-induced cardiomyocyte injury in an ErbB2-dependent manner. In summary, our study provides the first evidence that metformin protects cardiomyocyte against ethanol-induced oxidative stress and apoptosis by activating ErbB2-mediated AKT/Nrf2 signaling. Thus, metformin may be a potential novel treatment approach for alcoholic cardiomyopathy.

Novel mutant KRAS addiction signature predicts response to the combination of ERBB and MEK inhibitors in lung and pancreatic cancers

KRAS mutations are prevalent in pancreatic and lung cancers, but not all mutant (mt) KRAS tumors are addicted to mt KRAS. Here, we discovered a 30-gene transcriptome signature "KDS30" that encodes a novel EGFR/ERBB2-driven signaling network and predicts mt KRAS, but not NRAS or HRAS, oncogene addiction. High KDS30 tumors from mt KRAS lung and pancreatic cancer patients are enriched in genes upregulated by EGFR, ERBB2, mt KRAS or MEK. EGFR/ERBB2 (neratinib) and MEK (cobimetinib) inhibitor combination inhibits tumor growth and prolongs mouse survival in high, but not low, KDS30 mt KRAS lung and pancreatic xenografts, and is synergistic only in high KDS30 mt KRAS patient-derived organoids. Furthermore, mt KRAS high KDS30 lung and pancreatic cancer patients live significantly shorter lives than those with low KDS30. Thus, KDS30 can identify lung and pancreatic cancer patients whose tumors are addicted to mt KRAS, and predicts EGFR/ERBB2 and MEK inhibitor combination response.

Diffuse malignant peritoneal mesothelioma: A review.

Diffuse malignant peritoneal mesothelioma (DMPM) is an unusual and life-threatening locally invasive tumor. The morbidity and mortality of the disease are associated with progressive local effects in the abdominal cavity, such as abdominal distention, painful sensations, and early saturation with reduced oral intake, which eventually lead to intestinal obstruction and cachexia. Computed tomography (CT) has been widely used as a first-line diagnostic tool for DMPM. In addition, the most sensitive immunohistochemical markers of DMPM include WT 1, D2-40, and calmodulin. This paradigm has altered with the advancements in the immunohistochemical analysis of BRCA1-Associated Protein 1 (BAP1) the lack of BAP1 expression shows the diagnosis of malignancy. DMPM is resistant to conventional chemotherapies. Therefore, the gold standard for the treatment of DMPM is the combination of cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). The overexpression of the phosphatidylinositol 3-kinase (PI3K)/AKT serine/threonine kinase 1 (AKT)/mammalian target of rapamycin (mTOR) signaling pathway drives the malignant phenotype of DMPM, thereby showing promising potential for the treatment of DMPM. The coordinated activities among multiple RTKs are directly involved in the biological processes of DMPM, suggesting that the combined inhibition of the PI3K and mTOR signaling pathways might be an effective measure. This treatment strategy can be easily implemented in clinical practice. However, the combined inhibition of ERBB1(HER1)/ERBB2 (HER2) and ERBB3 (HER3) requires further investigations. Thus, based on these, the discovery of novel targeted therapies might be crucial to improving the prognosis of DMPM patients.

QSAR Study, Molecular Docking, and Pharmacokinetic Analysis of Substituted Dihydropyrimidinone as ErbB2 Inhibitors

ErbB2 is considered to be the preferred dimerization partner among the ErbB receptor tyrosine kinase (RTK) family. They are implicated in the development and progression of several cancer types, majorly breast cancer-related maladies. In the current study, quantitative structure-activity relationship (QSAR), molecular docking approaches and prediction of pharmacokinetic properties were utilized to identify promising ErbB2 inhibitors from a series of dihydropyrimidinone (DHPM) derivatives. The results indicated that the QSAR models of ErbB2 inhibitory activity is robust and has a very good prediction capacity, as exhibited by the value of R which is 0.9194. Furthermore, 6 compounds were shortlisted with potentially high biological activity. These compounds were subjected to drug likeliness, molecular docking and pharmacokinetic evaluations which indicated that the compounds are orally bioavailable and exhibit suitable proficiency as ErbB2 antagonists.

Abstract B073: C-terminal Binding Protein (CtBP): a critical regulator of ErbB2/3 signaling in PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains among the most lethal of human cancers with a 5-year survival of ~10%, underscoring the need to identify new therapeutic vulnerabilities. Previously, we reported high expression of transcriptional co-regulators C-terminal binding proteins (CtBP) 1 and 2 in human PDAC, however their precise role in PDAC formation or progression remains unclear. Here we have studied PDAC tumor dependency on CtBPs for growth and metastasis, using an orthotopic syngeneic pancreatic tumor mouse model employing a mouse PDAC cell line (CKP) derived from a pancreatic tumor that formed in a mouse expressing mutated K-Ras in the setting of p53 deletion. CRISPR-based homozygous deletion of Ctbp2 in the parental mouse PDAC cell line (CKP CtBP2 KO) dramatically decreased orthotopic PDAC tumor growth, drastically reduced metastatic potential, and significantly prolonged survival. Interrogating differential gene expression of the orthotopic PDAC tumors from Ctbp2 WT vs Ctbp2 KO cohorts, we identified significant downregulation of the EGFR-superfamily receptor ErbB3 in Ctbp2 KO tumors, and further determined that CtBP2 regulates expression of both the ErbB2 and 3 EGFR superfamily genes in PDAC cells. We therefore hypothesized that CtBP regulation of physiologic ErbB2/3 signaling contributes, in part, to PDAC growth and metastasis. Indeed, we observed that Ctbp2 KO cells exhibited near complete loss of ErbB2/3 expression and exhibited severely attenuated activation of phospho-Akt after neuregulin stimulation of ErbB2/3, and furthermore, human PDAC cells also demonstrated CtBP dependence of ErbB3 expression. Our results suggest that a subset of PDAC tumors are dependent on physiologic ErbB2/3 signaling and could be targeted by pharmacologic inhibitors of ErbB2 and/or ErbB3. Providing proof of concept, the ErbB2-targeted multikinase inhibitor lapatinib effectively killed the ErbB3 expressing CKP PDAC cells, while CKP CtBP2 KO cells, where ErbB3 expression was extinguished, were resistant to lapatinib. Taken together, our data suggests that ErB2/3 targeted therapeutics can effectively target a critical PDAC dependency on physiologic ErbB2/3 signaling which is the result of CtBP’s oncogenic transcriptional program that drives PDAC tumor progression. Citation Format: Kranthi Kumar Chougoni, Haemin Park, Priyadarshan K. Damle, Martin Michael Dcona, Barbara Szomju, Mikhail G. Dozmorov, Michael Idowu, Steven R. Grossman. C-terminal Binding Protein (CtBP): a critical regulator of ErbB2/3 signaling in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B073.

Abstract A072: Strategies to improve KRAS G12D inhibitor (MRTX1133) therapy in pancreatic ductal adenocarcinoma

Abstract Background: More than 90% of pancreatic ductal adenocarcinomas (PDAC) harbor oncogenic KRAS, with KRAS G12D being the predominant mutation. Recently, a novel KRAS G12D inhibitor, MRTX-1133, has been developed, potentially revolutionizing PDAC treatment. However, clinical studies on KRAS G12C inhibitors suggest that there may be intrinsic resistance to KRAS inhibitors (KRASi) and acquired resistance can develop rapidly in patients. Moreover, our preliminary data suggest that acquired resistance mechanisms to chemotherapy can also confer partial resistance to KRASi. We hypothesize that effective killing of tumor cells with KRASi will require a rationally designed combinatorial treatment. Methods: We tested chemotherapy and targeted pathway inhibitors which can inhibit signaling upstream and downstream of KRAS to identify drugs that synergize with MRTX-1133. We treated resected human PDAC and mouse tumors from Kras-G12D; P53-R172H; PDX-Cre (KPC) prepared as organotypic slice cultures to test our identified drug combinations. We orthotopically inoculated KPC PDAC cells and treated mice with vehicle, single-agent drugs (MRTX1133 or Afatinib), or combination therapy. We established MRTX-1133-resistant (MRTXR) models in human SUIT2 cells, human organoids and mouse KPC cell lines to study the mechanisms of KRASi resistance. We performed RNA-seq and Reverse Phase Protein Array in these resistance models to identify pathways differentially activated during resistance development. Results: We found that irreversible ErbB inhibitors, Afatinib and Neratinib, had the greatest synergy with MRTX-1133 in parental and in MRTXR cells. We found that combination therapy with MRTX-1133 and Afatinib decreased the number of proliferating cells compared to the control or single-agent treatment in the human and mouse slice cultures. Combination therapy with MRTX1133 and Afatinib significantly reduced tumor growth in mice compared to the vehicle or single-agent controls. We also found that resistant cells demonstrated increased EGFR and HER2 phosphorylation compared to parental cells and that the MRTX-1133 and Afatinib drug combo synergistically decreased EGFR and HER2 phosphorylation. We identified and mapped the differentially expressed pathways and genes involved in KRASi resistance using RNA-seq and RPPA analyses. Conclusion: We identified a likely pathway of resistance to novel KRAS G12D inhibitors, and we demonstrate that this resistance can be effectively targeted using existing therapeutics targeting the ErbB pathway. Citation Format: Kevin Christian M. Gulay, Edgar Esparza, Xinlian Zhang, Evangeline S. Mose, Jonathan Weitz, Minya Pu, Karen Messer, Andrew M. Lowy, Herve Tiriac. Strategies to improve KRAS G12D inhibitor (MRTX1133) therapy in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A072.

Alteration in tyrosine phosphorylation of cardiac proteome and EGFR pathway contribute to hypertrophic cardiomyopathy

Alterations of serine/threonine phosphorylation of the cardiac proteome are a hallmark of heart failure. However, the contribution of tyrosine phosphorylation (pTyr) to the pathogenesis of cardiac hypertrophy remains unclear. We use global mapping to discover and quantify site-specific pTyr in two cardiac hypertrophic mouse models, i.e., cardiac overexpression of ErbB2 (TgErbB2) and α myosin heavy chain R403Q (R403Q-αMyHC Tg), compared to control hearts. From this, there are significant phosphoproteomic alterations in TgErbB2 mice in right ventricular cardiomyopathy, hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM) pathways. On the other hand, R403Q-αMyHC Tg mice indicated that the EGFR1 pathway is central for cardiac hypertrophy, along with angiopoietin, ErbB, growth hormone, and chemokine signaling pathways activation. Surprisingly, most myofilament proteins have downregulation of pTyr rather than upregulation. Kinase-substrate enrichment analysis (KSEA) shows a marked downregulation of MAPK pathway activity downstream of k-Ras in TgErbB2 mice and activation of EGFR, focal adhesion, PDGFR, and actin cytoskeleton pathways. In vivo ErbB2 inhibition by AG-825 decreases cardiomyocyte disarray. Serine/threonine and tyrosine phosphoproteome confirm the above-described pathways and the effectiveness of AG-825 Treatment. Thus, altered pTyr may play a regulatory role in cardiac hypertrophic models.