pan-ErbB Tyrosine Kinase Inhibitor 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.

ErbB signalling is a potential therapeutic target for vascular lesions with fibrous component.

Sporadic venous malformation (VM) and angiomatosis of soft tissue (AST) are benign, congenital vascular anomalies affecting venous vasculature. Depending on the size and location of the lesion, symptoms vary from motility disturbances to pain and disfigurement. Due to the high recurrence of the lesions, more effective therapies are needed. As targeting stromal cells has been an emerging concept in anti-angiogenic therapies, here, by using VM/AST patient samples, RNA-sequencing, cell culture techniques, and a xenograft mouse model, we investigated the crosstalk of endothelial cells (EC) and fibroblasts and its effect on vascular lesion growth. We report, for the first time, the expression and secretion of transforming growth factor A (TGFA) in ECs or intervascular stromal cells in AST and VM lesions. TGFA induced secretion of vascular endothelial growth factor (VEGF-A) in paracrine fashion, and regulated EC proliferation. Oncogenic PIK3CA variant in p.H1047R, a common somatic mutation found in these lesions, increased TGFA expression, enrichment of hallmark hypoxia, and in a mouse xenograft model, lesion size, and vascularization. Treatment with afatinib, a pan-ErbB tyrosine-kinase inhibitor, decreased vascularization and lesion size in a mouse xenograft model with ECs expressing oncogenic PIK3CA p.H1047R variant and fibroblasts. Based on the data, we suggest that targeting of both intervascular stromal cells and ECs is a potential treatment strategy for vascular lesions having a fibrous component. Academy of Finland, Ella and Georg Ehnrooth foundation, the ERC grants, Sigrid Jusélius Foundation, Finnish Foundation for Cardiovascular Research, Jane and Aatos Erkko Foundation, GeneCellNano Flagship program, and Department of Musculoskeletal and Plastic Surgery, Helsinki University Hospital.

Abstract P6-12-06: Neratinib vs. Trastuzumab plus Ribociclib in ERBB2-positive breast cancer: Preclinical mechanistic efficacy study

Abstract Background: Despite the wide-ranging clinical success of human epidermal growth factor receptor 2 (HER2)-directed therapies, many HER2-positive breast cancer patients eventually progress because of the development of primary or acquired resistance. PIK3CA is found often mutated in breast cancer (>30%, TCGA dataset) and responsible for HER2-directed treatment failure. Purpose: Inhibition of pan-tyrosine kinases of HER-receptors along with the blockade of estrogen receptor (ER) prevents the activation of downstream effectors and crosstalk between HER2 and ER, leading to tumor cell death. Similarly, the combined inhibition of HER2-receptor signaling with cell cycle arrest leads to efficient tumor cell apoptosis. Here, we evaluate the mechanistic efficacy and comparability of neratinib (N) (an irreversible pan-ERBB tyrosine kinase inhibitor) in combination with fulvestrant (F) against ribociclib (R) plus trastuzumab (T) in HER2-amplified breast cancer cells. Methods: HER2+ breast cancer cell lines with Rb- wild-type- BT474 (ER+), SKBR3 (ER-), and MDA-MB453 (ER-, PIK3CA mutated [H1047R]) were used for the study. Cells were treated with N+F (F added in ER+ cell line only) or R or T as a single-agent or combination and assessed for real-time proliferation, 3D ON-TOP assay, changes in mitochondrial potential, and apoptosis. Cells treated with R and/or T were additionally examined for cell cycle arrest and changes in CDK4 mRNA transcription. Baseline CCND1 mRNA transcripts were quantified by RT-qPCR. Immunohistochemistry was used to assess baseline BCL2 expression in HER2+ tumor microarrays (TMA). Western blot was used to evaluate the effects on key downstream signaling proteins in response to the above treatment or combination. Results: High CCND1 expression was found in HER2+ cell lines that show promise for CDK4/6 inhibition. High BCL2 expression was found in HER2+ TMA that confirmed the natural resistance to programmed cell death. BT474 and MDA-MB453 were highly sensitive to N (+F), and high growth inhibition was evident at lower doses (< 20 nM). SKBR3 was comparatively less sensitive to N monotherapy and required dosing >160 nM to induce marked cell death. BT474 and MDA-MB453 had pronounced cytostatic responses to R or T+R, while a moderate response was observed in SKBR3. Substantial reduction in CDK4 transcription was noted following R or R+T treatment in MDA-MB453/BT474 but not in SKBR3. Apoptosis assays confirmed enhanced cell death with the R+T combination in BT474 and MDA-MB453 but not in SKBR3. Inhibition of key downstream oncogenic signaling (p-AKT/p-S6RP/p-ERK) was more evident with N compared to R, T, or R+T combination. Attenuated p-Rb expression (Ser 780 & Ser 807/811) was detected in all cell lines from R administration, indicating interruption in cell cycle progression. Conclusion: Mechanistically, N+F was superior to T+R in terms of inhibition of HER2 and its downstream signaling in the HER2+/ER+ BC model. N+F induced significantly more apoptosis and inhibited cell proliferation compared to T+R. Additionally, N monotherapy was highly effective in HER2-amplified/ER-negative breast cancer cells with PIK3CA mutation. Citation Format: Nischal Koirala, Jennifer Aske, Xiaoqian Lin, Nandini Dey, Pradip De. Neratinib vs. Trastuzumab plus Ribociclib in ERBB2-positive breast cancer: Preclinical mechanistic efficacy study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-06.

<i>NRG1</i> fusion-driven tumors: biology, detection, and the therapeutic role of afatinib and other ErbB-targeting agents

ErbB-targeted treatments, such as afatinib, a pan-ErbB tyrosine kinase inhibitor, are potential therapeutic strategies to address unmet treatment needs in patients harboring NRG1 fusions. A review article published in Annals of Oncology discusses the biology, detection, and incidence of NRG1 fusions, and the therapeutic role of afatinib and other ErbB-targeted agents in patients with cancer harboring NRG1 fusions. This Publication Perspective video summarizes the review article on behalf of Dr Stephen Liu (Georgetown University Medical Center, Washington, D.C., USA) and his co-authors.

The Pan-ErbB tyrosine kinase inhibitor afatinib inhibits multiple steps of the mammarenavirus life cycle

The mammarenavirus Lassa virus (LASV) causes a life-threatening acute febrile disease, Lassa fever (LF). To date, no US Food and Drug Administration (FDA)-licensed medical countermeasures against LASV are available. This underscores the need for the development of novel anti-LASV drugs. Here, we screen an FDA-approved drug library to identify novel anti-LASV drug candidates using an infectious-free cell line expressing a functional LASV ribonucleoprotein (vRNP), where levels of vRNP-directed reporter gene expression serve as a surrogate for vRNP activity. Our screen identified the pan-ErbB tyrosine kinase inhibitor afatinib as a potent inhibitor of LASV vRNP activity. Afatinib inhibited multiplication of lymphocytic choriomeningitis virus (LCMV) a mammarenavirus closely related to LASV. Cell-based assays revealed that afatinib inhibited multiple steps of the LASV and LCMV life cycles. Afatinib also inhibited multiplication of Junín virus vaccine strain Candid#1, indicating that afatinib can have antiviral activity against a broad range of human pathogenic mammarenaviruses.

1172P High activity of poziotinib in G778_P780dup HER2 exon 20 insertion mutations in non-small cell lung cancer (NSCLC)

Poziotinib is an oral pan-ErbB tyrosine kinase inhibitor (TKI) with demonstrated clinical activity and an expected safety profile. Patients with NSCLC harboring HER2 exon 20 insertion mutations (ex20ins) have poor outcomes and lack approved targeted treatments. The G778_P780dup (G778) mutation is one of the most common, representing 9-19% of HER2 ex20ins in NSCLC. Previous studies have reported an overall response rate (ORR) ranging from 10-60% with TKIs in patients with G778. Here we report the effects of poziotinib in patients harboring the HER2 ex20ins G778 mutation in a relatively large population of treatment-naïve (C4) and previously treated (C2) patients with NSCLC.

1685P Oncogenic activity of recurrent ERBB4 mutations and their sensitivity to neratinib

Hundreds of somatic ERBB4 (HER4) mutations have been described in cancer tissues with very limited information available about their functional significance. Understanding the activity of ERBB4 mutations is needed to assess the relevance of targeting ERBB4 in human cancers with matched therapies, such as neratinib. Neratinib is an irreversible pan-ERBB tyrosine kinase inhibitor that potently inhibits ERBB4, and is currently approved for early stage and metastatic HER2+ breast cancers, and is under clinical evaluation for EGFR-, ERBB2-, and ERBB4-mutant cancers, including in the SUMMIT clinical trial (NCT01953926).

Pyrotinib monotherapy or pyrotinib in combination with capecitabine could significantly prolong progression-free survival and overall survival in patients with HER2-positive metastatic breast cancer.

1034 Background: In the NALA phase III trial, irreversible pan-ErbB tyrosine kinase inhibitor (TKI) neratinib plus capecitabine demonstrated significant improvement in the progression-free survival (PFS), but no significant benefit in overall survival (OS) compared to lapatinib plus capecitabine. However, another TKI pyrotinib plus capecitabine showed significant benefits in PFS and a trend of OS benefits in the PHOEBE phase III study. But in general, current data on survival of irreversible TKIs in patients with HER2-positive metastatic breast cancer (MBC) was limited. Thus, we performed a pooled analysis of individual patient data from the phase I to III trials in HER2-positive MBC patients receiving pyrotinib or pyrotinib combined with capecitabine, to provide a cumulative assessment on long-term outcomes of irreversible TKI. Methods: Individual patient data was collected and analyzed from the phase I trial for pyrotinib and pyrotinib plus capecitabine, the Pivotal phase II trial and the PHOEBE phase III trials that enrolled patients in China National Cancer Center. Next-generation sequencing was performed on circulating tumor DNA for predictive biomarkers in the phase I trial. Results: Between August 2013 and October 2018, a total of 120 patients were assigned to received pyrotinib (n = 38), pyrotinib plus capecitabine (n = 53) and lapatinib plus capecitabine (n = 29) across the above four trials. The median follow-up duration for OS was 73.6 months (95% CI:69.9-77.3 months). The estimated median PFS was 8.2 months (95% CI:5.6-10.9 months) in the pyrotinib monotherapy group and 22.0 months (95% CI:13.2-30.8 months) in the pyrotinib plus capecitabine group ( P= 0.002), while the median OS was 27.1 months (95% CI: 21.6-32.5 months) in the pyrotinib monotherapy group and 44.5 months (95% CI: 30.8-58.1 months) in the pyrotinib plus capecitabine group ( P= 0.065). In this pooled analysis, pyrotinib 400mg in combination with capecitabine, recommended for phase II and III trials, had significantly longer PFS (22.0 vs 6.9 months, P＜0.001) and OS (59.9 vs 31.2 months, P= 0.035) than lapatinib plus capecitabine. Analysis of all genetic alterations in baseline blood samples suggested that the patients harbored concomitant mutations in HER2-related signaling network (including HER2 bypass signaling pathway, PI3K/Akt/mTOR pathway and TP53) were observed with significantly poorer PFS and OS compared to none or one genetic alteration (median PFS, 7.3 vs. 26.1 months, P= 0.003; median OS, 25.1 vs. 48.0 months, P= 0.013). Conclusions: This pooled analysis based on phase I to III trials revealed promising PFS and OS was achieved in pyrotinib and pyrotinib plus capecitabine. Concomitant mutations in HER2-related signaling network may be a potential efficacy and prognosis biomarker for pyrotinib in HER2-positive MBC. Clinical trial information: NCT01937689,NCT02361112,NCT02422199,NCT03080805.

Efficacy of afatinib in patients with advanced/metastatic solid tumors harboring NRG1 gene fusions: A novel, prospective real-world outcomes study based on single-patient protocol data.

TPS3180 Background: Oncogenic neuregulin 1 ( NRG1) gene fusions occur in ̃0.2% of solid tumors overall and in up to 31% of cases of invasive mucinous lung adenocarcinoma [Laskin et al. Ann Oncol. 2020;31(12):1693–1703; Cadranel et al. Oncologist. 2021;26(1):7–16]. NRG1 fusion proteins provide an extracellular anchor for the epidermal growth factor (EGF) domain of NRG1 to bind to ErbB3 (HER3), leading to HER3 heterodimerization and activation of downstream signaling pathways, resulting in oncogenesis. Afatinib, an irreversible pan-ErbB tyrosine kinase inhibitor, represents a potential treatment for NRG1-fusion positive ( NRG1+) tumors. This study aims to examine the safety and efficacy of afatinib in patients with NRG1+ solid tumors, for which no authorized targeted therapy exists. Methods: This prospective, decentralized, US study (NCT05107193) will include 40 evaluable patients aged ≥18 years. Participating molecular test providers across the USA will identify eligible fusions in the course of routine diagnostic assays. When a patient with an NRG1 fusion is identified, participating test providers will notify the treating physician of the study as a treatment option for the patient. Patients’ primary oncologists will then contact the trial sponsor to confirm patient eligibility. Once approved by the central Institutional Review Board, patients will receive afatinib on a single-patient protocol basis, until disease progression or treatment is no longer tolerated. The recommended dosage per SmPC is 40 mg orally QD. Patients will be screened and enrolled into the study at their existing point-of-care setting. Inclusion criteria include a histologically or cytologically confirmed diagnosis of an advanced, unresectable/metastatic, non-hematologic malignancy with an NRG1 fusion, evaluable per RECIST 1.1. Any coding gene as the NRG1 fusion partner is permitted. Fusion status will be confirmed prospectively by a contracted molecular test provider. Exclusion criteria include prior systemic anti-cancer therapy or investigational drug within 14 days or 5 half-lives (whichever is shorter) of the start of afatinib treatment; an actionable driver mutation other than NRG1 fusion for which FDA-approved targeted therapy is available; and prior treatment with an ErbB-targeted therapy. The primary endpoint of the study is confirmed objective response (OR) by independent central review per RECIST 1.1, defined as best overall response of either complete response or partial response and analysed as the proportion of patients with an OR. The key secondary endpoint is duration of response, defined as the time from the first documented OR to progression or death. Secondary endpoints include time to OR and disease control per investigator assessment. Safety will also be assessed. The study is open for recruitment. Clinical trial information: NCT05107193.

Antibiotic treatment targeting gram negative bacteria prevents neratinib-induced diarrhea in rats

BackgroundNeratinib is a pan-ErbB tyrosine kinase inhibitor used for extended adjuvant treatment of HER2-positive breast cancer. Diarrhea is the main adverse event associated with neratinib treatment. We aimed here to determine whether antibiotic-induced gut microbial shifts altered development of neratinib-induced diarrhea. MethodsFemale Albino Wistar rats (total n = 44) were given antibiotics (vancomycin, neomycin, or a cocktail of vancomycin, neomycin and ampicillin) in drinking water for four weeks, and then treated daily with neratinib (50 mg/kg) for 28 days. Diarrhea, along with markers of gastrointestinal damage and microbial alterations were measured by histopathology and 16S sequencing, respectively. ResultsRats treated with vancomycin or neomycin had significantly lower levels of diarrhea than rats treated with neratinib alone. In the distal ileum, neratinib was associated with a statistically significant increase in histological damage in all treatment groups expect the antibiotic cocktail. Key features included villous blunting and fusion and some inflammatory infiltrate. Differences in microbial composition at necropsy in vehicle control, neratinib and neratinib + neomycin groups, were characterized by a neratinib-induced increase in gram-negative bacteria that was reversed by neomycin. Neomycin shifted bacterial composition so that Blautia become the dominant genus. ConclusionsNarrow spectrum antibiotics reduced neratinib-induced diarrhea. This suggests that the microbiome may play a key role in the development and prolongation of diarrhea following neratinib treatment, although further research is required to understand the key bacteria and mechanisms by which they reduce diarrhea, as well as how this may impact presentation of diarrhea in clinical cohorts.

Abstract P05-02: A phase 1b study evaluating the combination of sotorasib, a KRASG12C inhibitor, and afatinib, a pan-ErbB tyrosine kinase inhibitor, in advanced KRAS p.G12C mutated non-small cell lung cancer (NSCLC)

Abstract Background: Sotorasib, a specific, irreversible KRASG12C inhibitor, was recently FDA-approved for adults with KRAS p.G12C-mutated locally advanced or metastatic NSCLC who received prior systemic therapy. The multi-arm phase 1b CodeBreaK101 master study is designed to evaluate the safety and tolerability of sotorasib in combination with both targeted and non-targeted therapies. Here we report the first safety and interim efficacy of sotorasib and afatinib, a pan-ErbB tyrosine kinase inhibitor, that had displayed synergistic antitumor activity in vitro. Methods: Patients with advanced KRAS p.G12C mutated NSCLC who had disease progression on prior therapies, including KRASG12C inhibitors, were enrolled in this dose exploration/expansion study. Patients were treated with 960 mg sotorasib QD and afatinib (20 mg or 30mg QD). Primary endpoint was safety/tolerability. Secondary endpoint was efficacy, including objective response rate (ORR) and disease control rate (DCR) per RECIST 1.1. Results: Based on a July 12, 2021 snapshot, 33 pts with NSCLC (median age: 65.0 yrs) were enrolled. Ten pts received 20 mg afatinib/960 mg sotorasib QD (cohort 1) and 23 pts received 30 mg afatinib/960 mg sotorasib QD (cohort 2). Across both cohorts, the median number of prior therapies was 2 (range 0-7; 66.7% ≥ 2 prior lines) and 5 pts (15.2%) received prior sotorasib. The most common treatment-related adverse events (TRAEs) included diarrhea (23 pts [69.7%], 7 pts [21.2%] grade 3), nausea (7 pts [21.2%], all grade ≤ 2), and vomiting (6 pts [18.2%], all grade ≤ 2). Grade ≥ 3 TRAEs occurred in 30% pts within each dose cohort, with diarrhea being the most common. Eight pts (24.2%) discontinued sotorasib and/or afatinib due to an AE, with diarrhea leading to discontinuation in 5 pts. Median treatment duration of sotorasib/afatinib combination was 64.0 days (Q1, 29.0; Q3, 128.0). For cohort 1 (N=10 that includes 4 pts who received prior sotorasib), the ORR was 20.0% and DCR was 70.0% (2-confirmed partial response (PR), 5-stable disease (SD), 1-progressive disease (PD), 2-not available); among the KRASG12C inhibitor-naïve patients, the ORR was 33.3%. For cohort 2 (N=23 that includes 1 pt who received prior sotorasib), the ORR was 34.8% and DCR was 73.9% (8-confirmed PR, 9-SD, 4-PD, 2-not available). Among 5 pts receiving prior sotorasib, 3 had SD, 1 PD, and 1 withdrew from study due to an AE prior to any scan. EGFR co-mutation at baseline was detected in 4 of 24 pts; 2 of 4 pts responded to combination therapy. Conclusions: In this first report of a combination of sotorasib and a pan-ErbB inhibitor, combining sotorasib with afatinib was feasible in a heavily pre-treated KRAS p.G12C mutated NSCLC population that included 15% who progressed on prior sotorasib. The AEs observed are not new or novel to either agent, with diarrhea being the most common. Investigation is ongoing to further explore this combination in an expansion cohort of the CodeBreaK101 master protocol. Citation Format: David Gandara, Kristen Marrone, Ramaswamy Govindan, Ferdinandos Skoulidis, Gregory Durm, Jeffrey Clarke, Richard Frank, John Krauss, Wendy Snyder, Tian Dai, Omar Mather, Paul Cifuentes, Antreas Hindoyan, Abraham Anderson, Timothy Burns. A phase 1b study evaluating the combination of sotorasib, a KRASG12C inhibitor, and afatinib, a pan-ErbB tyrosine kinase inhibitor, in advanced KRAS p.G12C mutated non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P05-02.

Simultaneous determination of dacomitinib and its major metabolite, O-desmethyl dacomitinib in human plasma by LC-MS/MS and its application to clinical testing in patients with non-small cell lung cancer

Dacomitinib, an irreversible pan-ErbB tyrosine kinase inhibitor targeting the human epidermal growth factor receptor, is used for the treatment of metastatic non-small cell lung cancer. To facilitate the investigations on its metabolism and other relevant studies, based on high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS), a rapid and sensitive bioanalytical technique was established and fully validated for simultaneous quantification of dacomitinib and its main metabolite in human plasma. The plasma samples were treated with acetonitrile containing 0.1% formic acid and the liquid supernatant was collected, dried and dissolved in methanol-water-formic acid (200:800:1, v/v) before injection. The chromatographic separation was performed on an ACE Excel C18 column (2.1 mm × 50.0 mm, i.d., 5 μm) by gradient elution with a mixture of buffer (5 mM ammonium acetate in 0.1% formic acid) and acetonitrile, serving as the mobile phase, with an overall run time of 4 min. Dacomitinib, O-desmethyl dacomitinib and IS were subsequently detected on an AB QTRAP 5500 mass spectrometer in positive ion and multiple reaction monitoring modes at the precursor-to-product transitions of m/z 470.4 → 385.0, m/z 456.0 → 370.9 and m/z 480.2 → 385.1, respectively. The accuracy and precision of determinations were guaranteed within the concentration ranges of 0.25–100 ng/mL for dacomitinib and 0.20–80 ng/mL for O-desmethyl dacomitinib. The intra- and inter-assay accuracy ranged from 92.00% to 104.50% and the intra- and inter-assay precision was less than 8.20% for each analyte. The method was validated and the relevant parameters, including selectivity, interference among analytes and internal standard, carry-over effect, dilution integrity, extraction recovery, matrix effect, and stability, all satisfied the requirements formulated by the US Food and Drug Administration and the European Medicines Agency. The clinical applicability of the fully-validated method was evaluated in medicated samples from patients on dacomitinib.

Population pharmacokinetic modeling of pyrotinib in patients with HER2-positive advanced or metastatic breast cancer

ObjectivePyrotinib, a new oral irreversible pan-ErbB tyrosine kinase inhibitor (TKI), has been approved in China for the treatment of HER2-positive advanced or metastatic breast cancer. This study aimed to conduct a population pharmacokinetics (PK) analysis of pyrotinib and to evaluate the impact of patient characteristics on pyrotinib's PK. MethodA total of 1152 samples, provided by 59 adult female patients from two phase I clinical trials, were analyzed by nonlinear mixed-effects modeling. Monte Carlo simulation was conducted to assess impact of covariates on the exposure to pyrotinib. ResultsThe PK of pyrotinib was adequately described by a one-compartment model with first-order absorption and elimination. Patient's age and total protein levels could affect pyrotinib's apparent volume of distribution, and concomitant use of montmorillonite could decrease the bioavailability of pyrotinib by 50.3%. No PK interactions were observed between capecitabine and pyrotinib. ConclusionIn this study, a population PK model of pyrotinib was developed to determine the influence of patient characteristics on the PK of pyrotinib. While patient age and total protein levels can significantly affect the apparent distribution volume of pyrotinib, the magnitude of the impact was limited, thus no dosage adjustment was recommended. Furthermore, concomitant use of montmorillonite for diarrhea needs to be taken with precaution.

NRG1 fusion-driven tumors: biology, detection, and the therapeutic role of afatinib and other ErbB-targeting agents.

Oncogenic gene fusions are hybrid genes that result from structural DNA rearrangements, leading to deregulated activity. Fusions involving the neuregulin-1 gene (NRG1) result in ErbB-mediated pathway activation and therefore present a rational candidate for targeted treatment. The most frequently reported NRG1 fusion is CD74-NRG1, which most commonly occurs in patients with invasive mucinous adenocarcinomas (IMAs) of the lung, although several other NRG1 fusion partners have been identified in patients with lung cancer, including ATP1B1, SDC4, and RBPMS. NRG1 fusions are also present in patients with other solid tumors, such as pancreatic ductal adenocarcinoma. In general, NRG1 fusions are rare across different types of cancer, with a reported incidence of <1%, with the notable exception of IMA, which represents ≈2%-10% of lung adenocarcinomas and has a reported incidence of ≈10%-30% for NRG1 fusions. A substantial proportion (≈20%) of NRG1 fusion-positive non-small-cell lung cancer cases are nonmucinous adenocarcinomas. ErbB-targeted treatments, such as afatinib, a pan-ErbB tyrosine kinase inhibitor, are potential therapeutic strategies to address unmet treatment needs in patients harboring NRG1 fusions.

Pathophysiology of neratinib-induced diarrhea in male and female rats: microbial alterations a potential determinant.

Neratinib is a potent irreversible pan-ErbB tyrosine kinase inhibitor, approved by the FDA for extended adjuvant treatment of HER2-positive breast cancer. Diarrhea is the most frequently observed adverse event with tyrosine kinase inhibitor therapy. In this study, we developed a reproducible model for neratinib-induced diarrhea in male and female rats. At first, male rats were treated with neratinib at 15, 30 or 50mg/kg or vehicle control via oral gavage for 28days (total n = 12). Secondly, we compared outcomes of male (n = 7) and female (n = 8) rats, treated with 50mg/kg neratinib. Rats treated with a 50mg/kg daily dose of neratinib had a reproducible and clinically relevant level of diarrhea and therefore was confirmed as an appropriate dose. Male rats treated with neratinib had significant changes to their gut microbiome. This included neratinib-induced increases in Ruminococcaceae (P = 0.0023) and Oscillospira (P = 0.026), and decreases in Blautia (P = 0.0002). On average, female rats experienced more significant neratinib-induced diarrhea (mean grade 1.526) compared with male rats (mean grade 1.182) (P < 0.0001). Neratinib caused a reduction in percentage weight gain after 28days of treatment in females (P = 0.0018) compared with vehicle controls. Females and males both showed instances of villus atrophy and fusion, most severely in the distal ileum. Serum neratinib concentration was higher in female rats compared to male rats (P = 0.043). A reproducible diarrhea model was developed in both female and male rats, which indicated that diarrhea pathogenesis is multifactorial, including anatomical disruption particularly evident in the distal ileum, and alterations in microbial composition.

Induction of autophagy enhances apoptotic cell death via epidermal growth factor receptor inhibition by canertinib in cervical cancer cells

BackgroundIt has been known epidermal growth factor receptor (EGFR) frequently overexpressed in cervical cancer. High levels of EGFR expression in their tumors leads to a poor prognosis and inhibition frequently induces autophagy in cancer cells. This study aimed to investigate whether EGFR inhibition by canertinib induces autophagy and this induction influence the effect of Palladium (Pd) (II) complex and 5-fluorouracil (5-FU) especially in nontoxic doses. MethodsCytotoxicity was evaluated by using SRB assay. Apoptosis, autophagy, and EGFR key markers were determined by flow cytometry, fluorescence staining, and immunoblotting. Colony formation, invasion, and wound healing assays were performed to investigate cell proliferation, invasion, and migration, respectively. ResultsBlocking EGFR by the pan-ErbB tyrosine kinase inhibitor canertinib inhibited cell growth of HeLa cervical cancer cells in combination with Pd(II) complex and 5-FU. Combination of canertinib and Pd(II) complex promotes autophagy and apoptosis of HeLa cancer cells via blockade of the PI3K/AKT and MAPK/ERK pathway, which leads to cervical cancer cell death. ROS accumulation and DNA damage were increased after combinatorial treatment which causes depolarization of the mitochondrial inner membrane and leads to apoptotic cell death. Canertinib combined with Pd(II) complex leads to inhibition of migration and invasion. ConclusionInhibition of EGFR signaling by canertinib in combination with Pd(II) complex promotes apoptosis and autophagy via blockade of the PI3K/AKT and MAPK/ERK. General significanceThe cytotoxic activity of Pd(II) complex and 5-FU on HeLa cells is mediated by EGFR inhibition and autophagy induction, leading to activation of mitochondrial apoptotic cell death.

Abstract OT2-07-09: A phase Ib study of neratinib, pertuzumab, and trastuzumab with paclitaxel in patients with metastatic and locally advanced breast cancer

Abstract Background: Neratinib, a potent irreversible pan-ErbB tyrosine kinase inhibitor that blocks signal transduction through HER1, HER2, and HER4, has demonstrated activity against metastatic HER2-positive breast cancer (HER2+ BC) in patients pretreated with trastuzumab. The FDA recently approved neratinib as an adjuvant treatment for HER2+ BC patients who have received trastuzumab for at least 1 year. Preclinical data demonstrate that trastuzumab-resistant BC cells remain sensitive to neratinib. Also, neratinib enhances responses to trastuzumab possibly by increasing trastuzumab's internalization, immune-mediated action, and other mechanisms. Taken together, these findings provide the rationale for adding neratinib to the standard of care combination of trastuzumab and pertuzumab with paclitaxel to enhance anti-HER2 efficacy in advanced HER2+ BC. Here, we report on the phase Ib portion of an ongoing phase Ib/II trial of this drug combination. Trial Design: Patients with metastatic or locally advanced HER2+ BC will be enrolled in the phase Ib portion of the trial. Neratinib is given orally in 3-week cycles. The initial neratinib dose of 80 mg daily is increased to 120, 160, and 200 mg daily after safety assessments of each dose level. Other agents are administered as per the standard of care. Patients continue therapy with per-protocol dose escalation and de-escalation according to toxicity until the maximum tolerated dose (MTD) of neratinib is reached. The target maximum dose-limiting toxicity rate is 20%. All patients receive 4 cycles of the combination therapy. If patients do not have disease progression or excessive toxicity, they may receive 2-4 additional cycles at the treating physician's discretion. During therapy, patients undergo blood tests every week and have clinical visits and restaging scans every 3 weeks. Because gastrointestinal toxicity, mainly diarrhea, is anticipated, patients receive prophylactic antidiarrheal medication (e.g., loperamide, budesonide) beginning with the first dose of neratinib. Eligibility Criteria: Eligible patients must have histologically confirmed metastatic or locally advanced HER2+ BC (BC may be inflammatory or non-inflammatory and have any hormone receptor status); an ECOG performance status score of 0 or 1; and adequate hematologic and organ function, including adequate cardiac function (as indicated by a left ventricle ejection fraction of ≥50%). Specific Aims: 1- To determine the MTD of neratinib in combination with paclitaxel, pertuzumab, and trastuzumab. 2- Pharmacodynamic markers will be measured on biologic specimens. Neratinib-induced changes in pEGFR and/or HER2 expression will be analyzed and compared between dose levels. Statistical Methods: The Bayesian modified toxicity probability interval is used to determine dose adjustment. Accrual: The target enrollment for the phase Ib cohort is 20 patients. The trial has enrolled 3 patients since its activation in January 2018. This trial is supported by Puma Biotechnology, Aggressive Breast Cancer Research Program Grant. Citation Format: Al-Awadhi A, Kono M, Marx A, Moseley T, Willey J, Sun H, Fu M, Whitman GJ, Valero V, Ueno NT, Lim B. A phase Ib study of neratinib, pertuzumab, and trastuzumab with paclitaxel in patients with metastatic and locally advanced breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT2-07-09.

Targeting neratinib-induced diarrhea with budesonide and colesevelam in a rat model.

Neratinib is an irreversible pan-ErbB tyrosine kinase inhibitor used for the extended adjuvant treatment of early-stage HER2-positive breast cancer. Its use is associated with the development of severe diarrhea in up to 40% of patients in the absence of proactive management. We previously developed a rat model of neratinib-induced diarrhea and found inflammation and anatomical disruption in the ileum and colon. Here we tested whether anti-diarrheal interventions, budesonide and colesevelam, can reduce neratinib-induced diarrhea and intestinal pathology. Rats were treated with 50mg/kg neratinib via oral gavage for 14 or 28days (total n = 64). Body weight and diarrhea severity were recorded daily. Apoptosis was measured using immunohistochemistry for caspase-3. Inflammation was measured via a multiplex cytokine/chemokine assay. ErbB levels were measured using PCR and Western Blot. Budesonide co-treatment caused rats to gain significantly less weight than neratinib alone from day 4 of treatment (P = 0.0418). Budesonide (P = 0.027) and colesevelam (P = 0.033) each reduced the amount of days with moderate diarrhea compared to neratinib alone. In the proximal colon, rats treated with neratinib had higher levels of apoptosis compared to controls (P = 0.0035). Budesonide reduced histopathological injury in the proximal (P = 0.0401) and distal colon (P = 0.027) and increased anti-inflammatory IL-4 tissue concentration (ileum; P = 0.0026, colon; P = 0.031) compared to rats treated with neratinib alone. In the distal ileum, while budesonide decreased ErbB1 mRNA expression compared to controls (P = 0.018) (PCR), an increase in total ErbB1 protein was detected (P = 0.0021) (Western Blot). Both budesonide and colesevelam show potential as effective interventions against neratinib-induced diarrhea.

Dacomitinib potentiates the efficacy of conventional chemotherapeutic agents via inhibiting the drug efflux function of ABCG2 in vitro and in vivo

BackgroundATP-binding cassette subfamily G member 2 (ABCG2), a member of the ABC transporter superfamily proteins, mediates multidrug resistance (MDR) by transporting substrate anticancer drugs out of cancer cells and decreasing their intracellular accumulation. MDR is a major hurdle to successful chemotherapy. A logical approach to overcome MDR is to inhibit the transporter. However, no safe and effective MDR inhibitor has been approved in the clinic.MethodsThe MTT assay was used to evaluate cell cytotoxicity and MDR reversal effect. Drug efflux and intracellular drug accumulation were measured by flow cytometry. The H460/MX20 cell xenograft model was established to evaluate the enhancement of anticancer efficacy of topotecan by dacomitinib in vivo. To ascertain the interaction of dacomitinib with the substrate binding sites of ABCG2, the competition of dacomitinib for photolabeling of ABCG2 with [125I]- iodoarylazidoprazosin (IAAP) was performed. Vanadate-sensitive ATPase activity of ABCG2 was measured in the presence of a range of different concentrations of dacomitinib to evaluate the effect of dacomitinib on ATP hydrolysis as the energy source of the transporter. A flow cytometry-based assay and western blotting were employed to study whether dacomitininb could inhibit the expression level of ABCG2. The mRNA expression levels of ABCG2 were analyzed by real-time quantitative RT-PCR. The protein expression level of AKT, ERK and their phosphorylations were detected by Western blotting.ResultsHere, we found that dacomitinib, an irreversible pan-ErbB tyrosine kinase inhibitor (TKI) in phase III clinical trial, could enhance the efficacy of conventional chemotherapeutic agents specifically in ABCG2-overexpressing MDR cancer cells but not in the parental sensitive cells. Dacomitinib was found to significantly increase the accumulation of ABCG2 probe substrates [doxorubicin (DOX),Rhodamine 123 (Rho 123) and Hoechst 33342] by inhibiting the transporter efflux function. Moreover, dacomitinib stimulated ABCG2 ATPase activity and competed with [125I]-IAAP photolabeling of ABCG2 in a concentration-dependent manner. However, dacomitinib did not alter ABCG2 expression at protein and mRNA levels or inhibit ErbB downstream signaling of AKT and ERK. Importantly, dacomitinib significantly enhanced the efficacy of topotecan in ABCG2-overexpressing H460/MX20 cell xenografts in nude mice without incurring additional toxicity.ConclusionsThese results suggest that dacomitinib reverses ABCG2-mediated MDR by inhibiting ABCG2 efflux function and increasing intracellular accumulation of anticancer agents. Our findings advocate further clinical investigation of combinations of dacomitinib and conventional chemotherapy in cancer patients with ABCG2-overexpressing MDR tumors.

Role of tyrosine kinase inhibitors in the treatment of pituitary tumours: from bench to bedside.

Treatment of aggressive pituitary tumours often yields suboptimal control of the tumour and confers significant morbidity. Lactotroph and corticotroph-derived tumours express ErbB receptors and ligands, and mutations in ubiquitin-specific protease 8 (USP8), which alters epidermal growth factor receptor (EGFR) degradation, have been implicated in Cushing disease pathogenesis. EGFR tyrosine kinase inhibitor (TKI) therapy has emerged as a potential new therapeutic approach for patients with aggressive prolactinomas and Cushing disease. Using EGFR or human epidermal growth factor receptor 2-driven prolactin (PRL) promoters, transgenic mice develop large tumours that respond to TKI inhibition. In human corticotroph primary cultures, treatment with the pan-ErbB TKI canertinib as well as the EGFR TKI gefitinib suppresses proopiomelanocortin mRNA. USP8 mutations, detected in up to two-thirds of Cushing disease, may underlie the increase in EGFR signalling in these tumours. Human prolactinomas have differential ErbB receptor expression associated with aggressive behaviour and data from an ongoing clinical trial suggest that resistant prolactinomas may respond to the EGFR TKI lapatinib. Preclinical and clinical models substantiate the role of the EGFR pathway in corticotroph and lactotroph adenomas. Although further study is needed, results to date suggest that targeting the ErbB pathway may be an effective therapeutic approach for patients with aggressive pituitary tumours.