QM/MM Simulations of Afatinib-EGFR Addition: The Role of ß-Dimethylaminomethyl Substitution.

Acrylamides are the most commonly used warheads of targeted covalent inhibitors (TCIs) directed at cysteines; however, the reaction mechanisms of acrylamides in proteins remain controversial, particularly for those involving protonated or unreactive cysteines. Using the combined semiempirical quantum mechanics (QM)/molecular mechanics (MM) free energy simulations, we investigated the reaction between afatinib, the first TCI drug for cancer treatment, and Cys797 in the EGFR kinase. Afatinib contains a ß-dimethylaminomethyl (ß-DMAM) substitution which has been shown to enhance the intrinsic reactivity and potency against EGFR for related inhibitors. Two hypothesized reaction mechanisms were tested. Our data suggest that Cys797 becomes deprotonated in the presence of afatinib, and the reaction proceeds via a classical Michael addition mechanism, with Asp800 stabilizing the ion-pair reactant state ß-DMAM+/C797- and the transition state of the nucleophilic attack. Our work elucidates an important structure-activity relationship of acrylamides in proteins.

Afatinib or Bevacizumab in combination with Osimertinib efficiently control tumor development in orthotopic murine models of non-small lung cancer.

Lung cancer is one of the most common and deadliest cancers. Preclinical models are essential to study new therapies and combinations taking tumor genetics into account. We have established cell lines expressing the luciferase gene from lines with varied genetic backgrounds, commonly encountered in patients with pulmonary adenocarcinoma. We have characterized these lines by testing their response to multiple drugs. Thus, we have developed orthotopic preclinical mouse models of NSCLC with very high engraftment efficiency. These models allow the easy monitoring of tumor growth, particularly in response to treatment, and of tumor cells dissemination in the body. We show that concomitant treatment with osimertinib (3rd generation tyrosine kinase inhibitor targeting mutated EGFR) and bevacizumab (anti-angiogenic targeting VEGF) can have a beneficial therapeutic effect on EGFR-mutated tumors. We also show that the addition of afatinib to osimertinib-treated tumors in escape leads to tumor growth inhibition. No such effect is observed with selumetinib or simvastatin. These preclinical mouse models therefore make it possible to test innovative therapeutic combinations and are also a tool of choice for studying resistance mechanisms.

Clinical efficacy and identification of factors confer resistance to afatinib (tyrosine kinase inhibitor) in EGFR-overexpressing esophageal squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) is reportedly overexpressed in most esophageal squamous cell carcinoma (ESCC) patients, but anti-EGFR treatments offer limited survival benefits. Our preclinical data showed the promising antitumor activity of afatinib in EGFR-overexpressing ESCC. This proof-of-concept, phase II trial assessed the efficacy and safety of afatinib in pretreated metastatic ESCC patients (n = 41) with EGFR overexpression (NCT03940976). The study met its primary endpoint, with a confirmed objective response rate (ORR) of 39% in 38 efficacy-evaluable patients and a median overall survival of 7.8 months, with a manageable toxicity profile. Transcriptome analysis of pretreatment tumors revealed that neurotrophic receptor tyrosine kinase 2 (NTRK2) was negatively associated with afatinib sensitivity and might serve as a predictive biomarker, irrespective of EGFR expression. Notably, knocking down or inhibiting NTRK2 sensitized ESCC cells to afatinib treatment. Our study provides novel findings on the molecular factors underlying afatinib resistance and indicates that afatinib has the potential to become an important treatment for metastatic ESCC patients.

Topical JAK inhibition ameliorates EGFR inhibitor-induced rash in rodents and humans.

Epidermal growth factor receptor inhibitors (EGFRis) are used to treat many cancers, but their use is complicated by the development of a skin rash that may be severe, limiting their use and adversely affecting patient quality of life. Most studies of EGFRi-induced rash have focused on the fully developed stage of this skin disorder, and early pathological changes remain unclear. We analyzed high-throughput transcriptome sequencing of skin samples from rats exposed to the EGFRi afatinib and identified that keratinocyte activation is an early pathological alteration in EGFRi-induced rash. Mechanistically, the induction of S100 calcium-binding protein A9 (S100A9) occurred before skin barrier disruption and led to keratinocyte activation, resulting in expression of specific cytokines, chemokines, and surface molecules such as interleukin 6 (Il6) and C-C motif chemokine ligand 2 (CCL2) to recruit and activate monocytes through activation of the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway, further recruiting more immune cells. Topical JAK inhibition suppressed the recruitment of immune cells and ameliorated the severity of skin rash in afatinib-treated rats and mice with epidermal deletion of EGFR, while having no effect on EGFRi efficacy in tumor-bearing mice. In a pilot clinical trial (NCT05120362), 11 patients with EGFRi-induced rash were treated with delgocitinib ointment, resulting in improvement in rash severity by at least one grade in 10 of them according to the MASCC EGFR inhibitor skin toxicity tool (MESTT) criteria. These findings provide a better understanding of the early pathophysiology of EGFRi-induced rash and suggest a strategy to manage this condition.

The ASCENT Trial: a phase 2 study of induction and consolidation afatinib and chemoradiation with or without surgery in stage III EGFR-mutant NSCLC.

BACKGROUND: The role of tyrosine kinase inhibitors (TKIs) in early-stage and metastatic oncogene-driven non-small cell lung cancer (NSCLC) is established, but it remains unknown how best to integrate TKIs with concurrent chemoradiotherapy (cCRT) in locally advanced disease. The phase 2 ASCENT trial assessed the efficacy and safety of afatinib and cCRT with or without surgery in locally advanced epidermal growth factor receptor (EGFR)-mutant NSCLC. PATIENTS AND METHODS: Adults ≥18 years with histologically confirmed stage III (AJCC 7th edition) NSCLC with activating EGFR mutations were enrolled at Mass General and Dana-Farber/Brigham Cancer Centers, Boston, Massachusetts. Patients received induction afatinib 40 mg daily for 2 months, then cisplatin 75 mg/m2 and pemetrexed 500 mg/m2 IV every 3 weeks during RT (definitive or neoadjuvant dosing). Patients with resectable disease underwent surgery. All patients were offered consolidation afatinib for 2 years. The primary endpoint was the objective response rate (ORR) to induction TKI. Secondary endpoints were safety, conversion to operability, progression-free survival (PFS), and overall survival (OS). Analyses were performed on the intention-to-treat population. RESULTS: Nineteen patients (median age 56 years; 74% female) were enrolled. ORR to induction afatinib was 63%. Seventeen patients received cCRT; 2/9 previously unresectable became resectable. Ten underwent surgery; 6 had a major or complete pathological response. Thirteen received consolidation afatinib. With a median follow-up of 5.0 years, median PFS and OS were 2.6 (95% CI, 1.4-3.1) and 5.8 years (2.9-NR), respectively. Sixteen recurred or died; 6 recurrences were isolated to CNS. The median time to progression after stopping consolidation TKI was 2.9 months (95% CI, 1.1-7.2). Four developed grade 2 pneumonitis. There were no treatment-related deaths. CONCLUSION: We explored the efficacy of combining TKI with cCRT in oncogene-driven NSCLC. Induction TKI did not compromise subsequent receipt of multimodality therapy. PFS was promising, but the prevalence of CNS-only recurrences and rapid progression after TKI discontinuation speak to unmet needs in measuring and eradicating micrometastatic disease.

Co-occurring EGFR p.E709X Mutation Mediates Primary Resistance to the Third-Generation EGFR-TKIs in EGFR p.G719X-Mutant Patients with Advanced NSCLC.

PURPOSE: The current National Comprehensive Cancer Network (NCCN) guidelines recommend afatinib or osimertinib as the preferred first-line treatment strategy for patients with advanced NSCLC harboring EGFR p.G719X mutation. However, in the absence of head-to-head trials comparing afatinib with osimertinib in EGFR p.G719X-mutant patients, it is unclear which regimen is the preferred treatment option. EXPERIMENTAL DESIGN: A large cohort of 4,228 treatment-naïve patients with lung cancer who underwent targeted next-generation sequencing (NGS) testing was screened for EGFR p.G719X mutation. A multicenter cohort involving 68 EGFR p.G719X-mutant patients with advanced NSCLC and NGS profiling was retrospectively enrolled to evaluate clinical responses to afatinib (n = 37) and the third-generation EGFR-TKIs (n = 31). Ba/F3 cells stably expressing the EGFR p.G719A mutation were created to investigate the response to EGFR-TKIs in vitro. RESULTS: Concurrent EGFR p.E709X mutations, being the most frequent co-occurring EGFR mutation in EGFR p.G719X-mutant NSCLC (∼30%), exerted a detrimental effect on outcomes in patients treated with third-generation EGFR-TKI [G719X/E709X vs. G719X; objective response rate (ORR): 0.00% vs. 47.62%, P < 0.001; mPFS: 7.18 vs. 14.2 months, P = 0.04, respectively]. Conversely, no significant difference was found in the treatment efficacy of afatinib between EGFR p.G719X/E709X and EGFR p.G719X patients (G719X/E709X vs. G719X; ORR: 71.43% vs. 56.67%, P = 0.99; mPFS: 14.7 vs. 15.8 months, P = 0.69, respectively). In vitro experiments elucidated a resistant drug sensitivity and poor inhibition of EGFR phosphorylation in Ba/F3 cells expressing EGFR p.G719A/E709K mutation upon the third-generation EGFR-TKI treatment. CONCLUSIONS: Co-occurring EGFR p.E709X mutation mediated primary resistance to the third-generation EGFR-TKIs in EGFR p.G719X-mutant patients but remained sensitive to afatinib. A personalized treatment strategy should be undertaken based on the coexisting EGFR p.E709X mutation status.

Single cell lineage tracing reveals clonal dynamics of anti-EGFR therapy resistance in triple negative breast cancer.

BACKGROUND: Most primary Triple Negative Breast Cancers (TNBCs) show amplification of the Epidermal Growth Factor Receptor (EGFR) gene, leading to increased protein expression. However, unlike other EGFR-driven cancers, targeting this receptor in TNBC yields inconsistent therapeutic responses. METHODS: To elucidate the underlying mechanisms of this variability, we employ cellular barcoding and single-cell transcriptomics to reconstruct the subclonal dynamics of EGFR-amplified TNBC cells in response to afatinib, a tyrosine kinase inhibitor (TKI) that irreversibly inhibits EGFR. RESULTS: Integrated lineage tracing analysis revealed a rare pre-existing subpopulation of cells with distinct biological signature, including elevated expression levels of Insulin-Like Growth Factor Binding Protein 2 (IGFBP2). We show that IGFBP2 overexpression is sufficient to render TNBC cells tolerant to afatinib treatment by activating the compensatory insulin-like growth factor I receptor (IGF1-R) signalling pathway. Finally, based on reconstructed mechanisms of resistance, we employ deep learning techniques to predict the afatinib sensitivity of TNBC cells. CONCLUSIONS: Our strategy proved effective in reconstructing the complex signalling network driving EGFR-targeted therapy resistance, offering new insights for the development of individualized treatment strategies in TNBC.

Deleterious association between proton pump inhibitor and protein kinase inhibitor exposure and survival for patients with lung cancer: A nationwide cohort study.

INTRODUCTION: Previous studies have identified an interaction between protein kinase inhibitors (PKIs) and proton pump inhibitors (PPIs) in patients with lung cancer. This type of interaction may reduce the efficacy of PKIs. However, the effect of PKI-PPI interaction on patient mortality remains controversial. This study set out to determine the impact of PKI-PPI interaction on overall survival for lung cancer patients. MATERIALS AND METHODS: This study was conducted using data from the French National Health Care Database from January 1, 2011 to December 31, 2021. We identified patients with: (i) an age equal to or greater than 18 years; (ii) lung cancer; and (iii) at least one reimbursement for one of the following drugs: erlotinib, gefitinib, afatinib and osimertinib. Patients were followed-up between the first date of PKI reimbursement and either December 31, 2021 or if they died, the date on which death occurred. The cumulative exposure to PPI duration during PKI treatment was calculated as the ratio between the number of concomitant exposure days to PKI and PPI and the number of exposure days to PKI. A survival analysis using a Cox proportional hazards model was then performed to assess the risk of death following exposure to a PKI-PPI interaction. RESULTS: 34,048 patients received at least one reimbursement for PKIs of interest in our study: 26,133 (76.8 %) were exposed to erlotinib; 3,142 (9.2 %) to gefitinib; 1,417 (4.2 %) to afatinib; and 3,356 (9.9 %) to osimertinib. Patients with concomitant exposure to PKI-PPI interaction during 20 % or more of the PKI treatment period demonstrated an increased risk of death (HR, 1.60 [95 % CI, 1.57-1.64]) compared to other patients. When this cut-off varied from 10 % to 80 %, the estimated HR ranged from 1.46 [95 % CI, 1.43-1.50] to 2.19 [95 % CI, 2.12-2.25]. DISCUSSION/CONCLUSION: In our study, an elevated risk of death was observed in patients exposed to PKI-PPI interaction. Finally, we were able to identify a dose-dependent effect for this interaction. This deleterious effect of osimertinib and PPI was revealed for the first time in real life conditions.

Romidepsin and Afatinib Abrogate Jak-Signal Transducer and Activator of Transcription Signaling and Elicit Synergistic Antitumor Effects in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphomas are mature lymphoid neoplasias resulting from the malignant transformation of skin-resident T-cells. A distinctive clinical feature of cutaneous T-cell lymphomas is their sensitivity to treatment with histone deacetylase inhibitors. However, responses to histone deacetylase inhibitor therapy are universally transient and noncurative, highlighting the need for effective and durable drug combinations. In this study, we demonstrate that the combination of romidepsin, a selective class I histone deacetylase inhibitor, with afatinib, an EGFR family inhibitor, induces strongly synergistic antitumor effects in cutaneous T-cell lymphoma models in vitro and in vivo through abrogation of Jak-signal transducer and activator of transcription signaling. These results support a previously unrecognized potential role for histone deacetylase inhibitor plus afatinib combination in the treatment of cutaneous T-cell lymphomas.

Real-world outcomes associated with afatinib use in patients with solid tumors harboring NRG1 gene fusions.

OBJECTIVES: Neuregulin-1 (NRG1) fusions may drive oncogenesis via constitutive activation of ErbB signaling. Hence, NRG1 fusion-driven tumors may be susceptible to ErbB-targeted therapy. Afatinib (irreversible pan-ErbB inhibitor) has demonstrated activity in individual patients with NRG1 fusion-positive solid tumors. This study collected real-world data on demographics, clinical characteristics, and clinical outcomes in this patient population. MATERIALS AND METHODS: In this retrospective, multicenter, non-comparative cohort study, physicians in the US-based Cardinal Health Oncology Provider Extended Network collected data from medical records of patients with NRG1 fusion-positive solid tumors who received afatinib (afatinib cohort) or other systemic therapies (non-afatinib cohort) in any therapy line. Objectives included demographics, clinical characteristics, and outcomes (overall response rate [ORR], progression-free survival [PFS], and overall survival [OS]). RESULTS: Patients (N = 110) with a variety of solid tumor types were included; 72 received afatinib, 38 other therapies. In the afatinib cohort, 70.8 % of patients received afatinib as second-line treatment and Eastern Cooperative Oncology Group performance status (ECOG PS) was 2-4 in 69.4 % at baseline. In the non-afatinib cohort, 94.7 % of patients received systemic therapy as first-line treatment and ECOG PS was 2-4 in 31.6 % at baseline. In the afatinib cohort, ORR was 37.5 % overall (43.8 % when received as first-line therapy); median PFS and OS were 5.5 and 7.2 months, respectively. In the non-afatinib cohort, ORR was 76.3 %; median PFS and OS were 12.9 and 22.6 months, respectively. CONCLUSION: This study provides real-world data on the characteristics of patients with NRG1 fusion-positive solid tumors treated with afatinib or other therapies; durable responses were observed in both groups. However, there were imbalances between the cohorts, and the study was not designed to compare outcomes. Further prospective/retrospective trials are required.

Assessing Efficacy of Afatinib toward Elastic Matrix Repair in Aortic Aneurysms.

Abdominal aortic aneurysm (AAA) is a critical, multifactorial cardiovascular disorder marked by localized dilatation of the abdominal aorta. A major challenge to countering the pathophysiology of AAAs lies in the naturally irreversible breakdown of elastic fibers in the aorta wall, which is linked to the poor elastogenicity of adult and diseased vascular smooth muscle cells (SMCs) and their impaired ability to assemble mature elastic fibers in a chronic proteolytic tissue milieu. We have previously shown that these are downstream effects of neutrophil elastase-induced activation of the epidermal growth factor receptor (EGFR) activity in aneurysmal SMCs. The novelty of this study lies in investigating the benefits of an EGFR inhibitor drug, afatinib (used to treat nonsmall cell lung cancer), for proelastogenic and antiproteolytic stimulation of aneurysmal SMCs. In in vitro cell cultures, we have shown that safe doses of 0.5 and 1 nM afatinib inhibit EGFR and p-extracellular signal-regulated kinases 1/2 protein expression by 50-70% and downstream elastolytic matrix metalloprotease 2 (MMP2) versus untreated control cultures. In addition, elastin production on a per cell basis was significantly upregulated by afatinib doses within the 0.1-1 nM dose range, which was further validated through transmission electron microscopy showing significantly increased presence of tropoelastin coacervates and maturing elastic fibers upon afatinib treatment at the above doses. Therefore, our studies for the first time demonstrate the therapeutic benefits of afatinib toward use for elastic matrix repair in small AAAs.

Prevalence and Therapeutic Targeting of High-Level ERBB2 Amplification in NSCLC.

INTRODUCTION: ERBB2 amplification in lung cancer remains poorly characterized. HER2 (encoded by ERBB2) is a transmembrane tyrosine kinase capable of ligand-independent dimerization and signaling when overexpressed, and a common cause of HER2 overexpression is ERBB2 amplification. Here, we evaluated the clinicopathologic and genomic characteristics of ERBB2-amplified NSCLC and explored a HER2 antibody-drug conjugate (ADC) therapeutic strategy. METHODS: Our institutional next-generation DNA sequencing data (OncoPanel) from 5769 NSCLC samples (5075 patients) were queried for cases having high-level ERBB2 amplification (≥6 copies). Clinical and demographic characteristics were extracted from the electronic medical records. Efficacy of the pan-ERBB inhibitor afatinib or HER2 ADCs (trastuzumab deruxtecan and trastuzumab emtansine) was evaluated in NSCLC preclinical models and patients with ERBB2 amplification. RESULTS: High-level ERBB2 amplification was identified in 0.9% of lung adenocarcinomas and reliably predicted overexpression of HER2. ERBB2 amplification events are detected in two distinct clinicopathologic and genomic subsets of NSCLC: as the sole mitogenic driver in tumors arising in patients with a smoking history or as a concomitant alteration with other mitogenic drivers in patients with a light or never smoking history. We further reveal that trastuzumab deruxtecan is effective therapy in in vitro and in vivo preclinical models of NSCLC harboring ERBB2 amplification and report two cases of clinical activity of an anti-HER2 ADC in patients who acquired ERBB2 amplification after previous targeted therapy. CONCLUSIONS: High-level ERBB2 amplification reliably predicts HER2 overexpression in patients with NSCLC, and HER2 ADC is effective therapy in this population.

Aspirin boosts the synergistic effect of EGFR/p53 inhibitors on lung cancer cells by regulating AKT/mTOR and p53 pathways.

The regimen of afatinib and vinorelbine has been used to treat breast or lung cancer cells with some limitations. Aspirin alone or in combination with other agents has shown unique efficacy in the treatment of cancer. We designed a preclinical study to investigate whether the triple therapy of aspirin, afatinib, and vinorelbine could synergistically inhibit the growth of p53 wild-type nonsmall cell lung cancer (NSCLC) cells. Three NSCLC cells A549, H460, and H1975 were selected to study the effect of triple therapy on cell proliferation and apoptosis. Compared to single agents, triple therapy synergistically inhibited the proliferation of lung cancer cells with combination index <1. Meanwhile, the therapeutic index of triple therapy was superior to that of single agents, indicating a balance between efficacy and safety in the combination of three agents. Mechanistic studies showed that triple therapy significantly induced apoptosis by decreasing mitochondrial membrane potential, increasing reactive oxygen species, and regulating mitochondria-related proteins. Moreover, epidermal growth factor receptor (EGFR) downstream signaling proteins including JNK, AKT, and mTOR were dramatically suppressed and p53 was substantially increased after NSCLC cells were exposed to the triple therapy. We provided evidence that the triple therapy of aspirin, afatinib and vinorelbine synergistically inhibited lung cancer cell growth through inactivation of the EGFR/AKT/mTOR pathway and accumulation of p53, providing a new treatment strategy for patients with p53 wild-type NSCLC.

Curcumin potentiates the ErbB receptors inhibitor Afatinib for enhanced antitumor activity in malignant mesothelioma.

Several attempts have been made to develop targeted therapies for malignant mesothelioma (MM), an aggressive tumour with a poor prognosis. In this study we evaluated whether Curcumin (CUR) potentiated the antitumor activity of the ErbB receptors inhibitor Afatinib (AFA) on MM, employing cell lines cultured in vitro and mice bearing intraperitoneally transplanted, syngeneic MM cells. The rationale behind this hypothesis was that CUR could counteract mechanisms of acquired resistance to AFA. We analysed CUR and AFA effects on MM cell growth, cell cycle, autophagy, and on the modulation of tumour-supporting signalling pathways.This study demonstrated that, as compared to the individual compounds, the combination of AFA + CUR had a stronger effect on MM progression which can be ascribed either to increased tumour cell growth inhibition or to an enhanced pro-apoptotic effect. These results warrant future studies aimed at further exploring the therapeutic potential of AFA + CUR-based combination regimens for MM treatment.

Dual Inhibition of KRASG12D and Pan-ERBB Is Synergistic in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a low survival rate. Recently, new drugs that target KRASG12D, a common mutation in PDAC, have been developed. We studied one of these compounds, MRTX1133, and found it was specific and effective at low nanomolar concentrations in patient-derived organoid models and cell lines harboring KRASG12D mutations. Treatment with MRTX1133 upregulated the expression and phosphorylation of EGFR and HER2, indicating that inhibition of ERBB signaling may potentiate MRTX1133 antitumor activity. Indeed, the irreversible pan-ERBB inhibitor, afatinib, potently synergized with MRTX1133 in vitro, and cancer cells with acquired resistance to MRTX1133 in vitro remained sensitive to this combination therapy. Finally, the combination of MRTX1133 and afatinib led to tumor regression and longer survival in orthotopic PDAC mouse models. These results suggest that dual inhibition of ERBB and KRAS signaling may be synergistic and circumvent the rapid development of acquired resistance in patients with KRAS mutant pancreatic cancer. SIGNIFICANCE: KRAS-mutant pancreatic cancer models, including KRAS inhibitor-resistant models, show exquisite sensitivity to combined pan-ERBB and KRAS targeting, which provides the rationale for testing this drug combination in clinical trials.

Natural Cyclophilin A Inhibitors Suppress the Growth of Cancer Stem Cells in Non-Small Cell Lung Cancer by Disrupting Crosstalk between CypA/CD147 and EGFR.

Non-small cell lung cancer (NSCLC) is a fatal malignant tumor with a high mortality rate. Cancer stem cells (CSCs) play pivotal roles in tumor initiation and progression, treatment resistance, and NSCLC recurrence. Therefore, the development of novel therapeutic targets and anticancer drugs that effectively block CSC growth may improve treatment outcomes in patients with NSCLC. In this study, we evaluated, for the first time, the effects of natural cyclophilin A (CypA) inhibitors, including 23-demethyl 8,13-deoxynargenicin (C9) and cyclosporin A (CsA), on the growth of NSCLC CSCs. C9 and CsA more sensitively inhibited the proliferation of epidermal growth factor receptor (EGFR)-mutant NSCLC CSCs than EGFR wild-type NSCLC CSCs. Both compounds suppressed the self-renewal ability of NSCLC CSCs and NSCLC-CSC-derived tumor growth in vivo. Furthermore, C9 and CsA inhibited NSCLC CSC growth by activating the intrinsic apoptotic pathway. Notably, C9 and CsA reduced the expression levels of major CSC markers, including integrin α6, CD133, CD44, ALDH1A1, Nanog, Oct4, and Sox2, through dual downregulation of the CypA/CD147 axis and EGFR activity in NSCLC CSCs. Our results also show that the EGFR tyrosine kinase inhibitor afatinib inactivated EGFR and decreased the expression levels of CypA and CD147 in NSCLC CSCs, suggesting close crosstalk between the CypA/CD147 and EGFR pathways in regulating NSCLC CSC growth. In addition, combined treatment with afatinib and C9 or CsA more potently inhibited the growth of EGFR-mutant NSCLC CSCs than single-compound treatments. These findings suggest that the natural CypA inhibitors C9 and CsA are potential anticancer agents that suppress the growth of EGFR-mutant NSCLC CSCs, either as monotherapy or in combination with afatinib, by interfering with the crosstalk between CypA/CD147 and EGFR.

Clinical Outcomes of Afatinib Versus Osimertinib in Patients With Non-Small Cell Lung Cancer With Uncommon EGFR Mutations: A Pooled Analysis.

BACKGROUND: The purpose of this analysis was to investigate the effectiveness of afatinib compared to that of osimertinib in patients with non-small cell lung cancer (NSCLC) who harbored uncommon epidermal growth factor receptor (EGFR) mutations. METHODS: A PubMed database-based literature review was conducted to retrieve related studies. Patients harboring EGFR mutations besides the deletion in exon 19 (19del) and point mutation of L858R were included in this analysis. The primary outcome events were the objective response rate (ORR) and progression-free survival (PFS). Propensity score matching (PSM) at a ratio of 1:1 was used between afatinib and osimertinib groups to control the confounding factors. Uncommon EGFR mutations were categorized into 4 groups: insertion in exon 20 (ex20ins), non-ex20ins single uncommon EGFR mutations, compound EGFR mutations that with 19del or L858R, and compound EGFR mutations without 19del or L858R. RESULTS: After PSM, 71 patients in either the afatinib or osimertinib group were matched. The afatinib group had an ORR of 60.6%, slightly higher than the osimertinib group's (50.3%), the difference was not statistically significant (P = .610). However, the afatinib group showed a significantly superior PFS benefit than the osimertinib group (11.0 vs. 7.0 months, P = .044). In addition, patients harboring non-ex20ins single uncommon EGFR mutations yield the best ORR and PFS, following treatment of either afatinib (ORR: 76.7%, mPFS: 14.1 months) or osimertinib (ORR: 68.8%, mPFS: 15.1 months). Moreover, there was no significant difference in terms of ORR or PFS between the cohort of patients treated with afatinib or osimertinib, regardless of whether or not the patients had brain metastases. CONCLUSIONS: Both afatinib and osimertinib displayed favorable clinical activities toward uncommon EGFR mutations. Afatinib showed a more profound and durable PFS benefit than osimertinib, although no efficacy advantage was observed.

Bruceine D and afatinib combination inhibits ovarian cancer cells proliferation and migration through DNA damage repair and EGFR pathway.

Owing to the high rates of relapse and migration, ovarian cancer (OC) has been recognized as the most lethal gynecological malignancy worldwide. The activity of the epidermal growth factor receptor (EGFR) signaling pathway is frequently associated with OC cell proliferation and migration. Despite this knowledge, inhibition of EGFR signaling in OC patients failed to achieve satisfactory therapeutic effects. In this study, we identified that bruceine D (BD) and EGFR inhibitor, afatinib, combination resulted in synergistic anti-OC effects. The results indicated that compared with one of both drugs alone, the combination of BD and afatinib slowed the DNA replication rate, inhibition of cell viability, and proliferation and clone formation. This resulted in cell cycle arrest and cell apoptosis. In addition, the combination of BD and afatinib possessed a stronger ability to inhibit the OC cell adhesion and migration than treatment with BD or afatinib alone. Mechanistically, the combined treatment triggered intense DNA damage, suppressed DNA damage repair, and enhanced the inhibition of the EGFR pathway. These results demonstrated that compared with each pathway inhibition, combined blocking of both DNA damage repair and the EGFR pathway appears to more effective against OC treatment. The results support the potential of BD and afatinib combination as a therapeutic strategy for OC patients.

NEP010, a novel compound with minor structural modification from afatinib, exhibited significantly improved antitumor activity.

Non-small cell lung cancer (NSCLC) is classified into many subclasses according to the specific kinase mutations. The most common mutation is epidermal growth factor receptor (EGFR) somatic mutation, which has promoted the development of several novel drugs (Tyrosine kinase inhibitors, TKIs). Although various TKIs are recommended as targeted therapy for NSCLC with EGFR mutations in NCCN guidelines, not all patients respond equally to the recommended TKIs, which lead to series of novel compound under development to satisfy actual clinical needs. Based on the structure of afatinib, a marketed drug recommended as the first-line therapy for patients with EGFR mutation, NEP010 was synthesized with structural modification. The antitumor efficacy of NEP010 on mouse tumor xenograft models with different EGFR mutations was determined. Results showed that with minor structural modifications on afatinib, the inhibitory effect of NEP010 on EGFR mutant tumors was significantly improved. Pharmacokinetics test was adopted, and compared with afatinib, the increased tissue exposure of NEP010 may be the potential factor responsible for the increased efficacy. Furthermore, tissue distribution test showed a high concentration of NEP010 in the lung which happens to be the target organ of NEP010 in clinical practice. In conclusion, data obtained suggested that NEP010 has an increased anti-tumor effect via improving pharmacokinetics, and may provide a potent therapeutic option for the patients with EGFR mutation of NSCLC in the future.

"From molecular to clinic": The pivotal role of CDC42 in pathophysiology of human papilloma virus related cancers and a correlated sensitivity of afatinib.

Background: Human papilloma virus (HPV)-related cancers are global health challenge. Insufficient comprehension of these cancers has impeded the development of novel therapeutic interventions. Bioinformatics empowered us to investigate these cancers from new entry points. Methods: DNA methylation data of cervical squamous cell carcinoma (CESC) and anal squamous cell carcinoma (ASCC) were analyzed to identify the significantly altered pathways. Through analyses integrated with RNA sequencing data of genes in these pathways, genes with strongest correlation to the TNM staging of CESC was identified and their correlations with overall survival in patients were assessed. To find a potential promising drug, correlation analysis of gene expression levels and compound sensitivity was performed. In vitro experiments were conducted to validate these findings. We further performed molecular docking experiments to explain our findings. Results: Significantly altered pathways included immune, HPV infection, oxidative stress, ferroptosis and necroptosis. 10 hub genes in these pathways (PSMD11, RB1, SAE1, TAF15, TFDP1, CORO1C, JOSD1, CDC42, KPNA2 and NUP62) were identified, in which only CDC42 high expression was statistically significantly correlated with overall survival (Hazard Ratio: 1.6, P = 0.045). Afatinib was then screened out to be tested. In vitro experiments exhibited that the expression level of CDC42 was upregulated in HaCaT/A431 cells transfected with HPV E6 and E7, and the inhibitory effect of afatinib on proliferation was enhanced after transfection. CDC42-GTPase-effector interface-EGFR-afatinib was found to be a stable complex with a highest ZDOCK score of 1264.017. Conclusion: We identified CDC42 as a pivotal gene in the pathophysiology of HPV-related cancers. The upregulation of CDC42 could be a signal for afatinib treatment and the mechanism in which may be an increased affinity of EGFR to afatinib, inferred from a high stability in the quaternary complex of CDC42-GTPase-effector interface-EGFR-afatinib.