Lapatinib antitumor effect is associated with PI3K and MAPK pathway: An analysis in human and canine prostate cancer cells.

The aberrant activation of HER2 has a pivotal role in bone metastasis implantation and progression in several tumor types, including prostate cancer (PC). Trastuzumab and other anti-HER2 therapies, such as lapatinib, have been used in human breast cancer HER2 positive. Although HER2 overexpression has been reported in PC, anti-HER2 therapy response has revealed conflicting results. We investigated the potential of lapatinib in inhibiting cell migration and inducing apoptosis in two human (LNCaP and PC3) and two canine PC cell lines (PC1 and PC2). Cell migration and apoptosis were evaluated by Annexin V/PI analysis after lapatinib treatment. The transcriptome analysis of all cell lines before and after treatment with lapatinib was also performed. We found increased apoptosis and migration inhibition in LNCaP cells (androgen-sensitive cell line), while PC1, PC2, and PC3 cells showed no alterations after the treatment. The transcriptome analysis of LNCaP and PC3 cell lines showed 158 dysregulated transcripts in common, while PC1 and PC2 cell lines presented 82. At the doses of lapatinib used, we observed transcriptional modifications in all cell lines. PI3K/AKT/mTOR pathway were enriched in human PC cells, while canine PC cells showed enrichment of tyrosine kinase antitumor response and HER2-related pathways. In canine PC cells, the apoptosis failed after lapatinib treatment, possibly due to the downregulation of MAPK genes. Prostate cancer cells insensitive to androgens may be resistant to lapatinib through PI3K gene dysregulation. The association of lapatinib with PI3K inhibitors may provide a more effective antitumor response and clinical benefits to PC patients.

Telangiectasias induced by combination tucatinib and ado-trastuzumab emtansine in a patient with metastatic breast cancer.

BACKGROUND: Tucatinib is a tyrosine kinase inhibitor currently used in salvage therapy for human epidermal growth factor receptor 2 (HER2)-positive breast and colorectal cancer. The use of tucatinib alone or in combination with ado-trastuzumab emtansine (T-DM1) in the treatment of advanced HER2-positive cancers is rapidly expanding. OBJECTIVE/METHODS: We report the case of a 66-year-old female who presented to the dermatology clinic with a one-year history of widespread telangiectasias that began after initiation of combination chemotherapy with tucatinib and T-DM1 for metastatic HER2-positive invasive ductal carcinoma. RESULTS: The patient's lesions regressed upon cessation of combination therapy and reappeared in the setting of tucatinib re-initiation, with gradual improvement over the following four months following electrocautery to the affected regions. CONCLUSIONS: We postulate that telangiectasias may be a previously unreported dermatologic side effect of combination treatment with tucatinib and T-DM1. Electrocautery is a safe and effective procedure to reduce the appearance of telangiectasias and improve patient satisfaction during chemotherapy.

Tumor growth-arrest effect of tetrahydroquinazoline-derivative human topoisomerase II-alpha inhibitor in HPV-negative head and neck squamous cell carcinoma.

Oral malignancies continue to have severe morbidity with less than 50% long-term survival despite the advancement in the available therapies. There is a persisting demand for new approaches to establish more efficient strategies for their treatment. In this regard, the human topoisomerase II (topoII) enzyme is a validated chemotherapeutics target, as topoII regulates vital cellular processes such as DNA replication, transcription, recombination, and chromosome segregation in cells. TopoII inhibitors are currently used to treat some neoplasms such as breast and small cells lung carcinomas. Additionally, topoII inhibitors are under investigation for the treatment of other cancer types, including oral cancer. Here, we report the therapeutic effect of a tetrahydroquinazoline derivative (named ARN21934) that preferentially inhibits the alpha isoform of human topoII. The treatment efficacy of ARN21934 has been evaluated in 2D cell cultures, 3D in vitro systems, and in chick chorioallantoic membrane cancer models. Overall, this work paves the way for further preclinical developments of ARN21934 and possibly other topoII alpha inhibitors of this promising chemical class as a new chemotherapeutic approach for the treatment of oral neoplasms.

Discovery of Novel 5,6-Dihydro-4H-pyrido[2,3,4-de]quinazoline Irreversible Inhibitors Targeting Both Wild-Type and A775_G776insYVMA Mutated HER2 Kinases.

HER2 mutations were seen in 4% of non-small-cell lung cancer (NSCLC) patients. Most of these mutations (90%) occur as an insertion mutation within the exon 20 frame, leading to the downstream activation of the PI3K-AKT and RAS/MAPK pathways. However, no targeted therapies have yet been approved worldwide. Here a novel series of highly potent HER2 inhibitors with a pyrido[2,3,4-de]quinazoline core were designed and developed. The derivatives with the pyrido[2,3,4-de]quinazoline core displayed superior efficacy of antiproliferation in BaF3 cells harboring HER2insYVMA mutation compared with afatinib and neratinib. Rat studies showed that 8a and 9a with the newly developed core have good pharmacokinetic properties with an oral bioavailability of 41.7 and 42.0%, respectively. Oral administration of 4a and 10e (30 mg/kg, QD) displayed significant antitumor efficacy in an in vivo xenograft model. We proposed promising strategies for the development of HER2insYVMA mutant inhibitors in this study.

Hit-to-lead optimization of 2-aminoquinazolines as anti-microbial agents against Leishmania donovani.

Visceral leishmaniasis is a potentially fatal disease caused by infection by the intracellular protist pathogens Leishmania donovani or Leishmania infantum. Present therapies are ineffective because of high costs, variable efficacy against different species, the requirement for hospitalization, toxicity and drug resistance. Detailed analysis of previously published hit molecules suggested a crucial role of 'guanidine' linkage for their efficacy against L. donovani. Here we report the design of 2-aminoquinazoline heterocycle as a basic pharmacophore-bearing guanidine linkage. The introduction of various groups and functionality at different positions of the quinazoline scaffold results in enhanced antiparasitic potency with modest host cell cytotoxicity using a physiologically relevant THP-1 transformed macrophage infection model. In terms of the ADME profile, the C7 position of quinazoline was identified as a guiding tool for designing better molecules. The good ADME profile of the compounds suggests that they merit further consideration as lead compounds for treating visceral leishmaniasis.

YES1 as a potential target to overcome drug resistance in EGFR-deregulated non-small cell lung cancer.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) such as gefitinib and osimertinib have primarily been used as first-line treatments for patients with EGFR-activating mutations in non-small cell lung cancer (NSCLC). Novel biomarkers are required to distinguish patients with lung cancer who are resistant to EGFR-TKIs. The aim of the study is to investigate the expression and functional role of YES1, one of the Src-family kinases, in EGFR-TKI-resistant NSCLC. YES1 expression was elevated in gefitinib-resistant HCC827 (HCC827/GR) cells, harboring EGFR mutations. Moreover, HCC827/GR cells exhibited increased reactive oxygen species (ROS) levels compared to those of the parent cells, resulting in the phosphorylation/activation of YES1 due to oxidation of the cysteine residue. HCC827/GR cells showed elevated expression levels of YES1-associated protein 1 (YAP1), NF-E2-related factor 2 (Nrf2), cancer stemness-related markers, and antioxidant proteins compared to those of the parent cells. Knockdown of YES1 in HCC827/GR cells suppressed YAP1 phosphorylation, leading to the inhibition of Bcl-2, Bcl-xL, and Cyclin D1 expression. Silencing YES1 markedly attenuated the proliferation, migration, and tumorigenicity of HCC827/GR cells. Dasatinib inhibited the proliferation of HCC827/GR cells by targeting YES1-mediated signaling pathways. Furthermore, the combination of gefitinib and dasatinib demonstrated a synergistic effect in suppressing the proliferation of HCC827/GR cells. Notably, YES1- and Nrf2-regulated genes showed a positive regulatory relationship in patients with lung cancer and in TKI-resistant NSCLC cell lines. Taken together, these findings suggest that modulation of YES1 expression and activity may be an attractive therapeutic strategy for the treatment of drug-resistant NSCLC.

Discovery of anticancer function of Febrifugine: Inhibition of cell proliferation, induction of apoptosis and suppression steroid synthesis in bladder cancer cells.

Bladder cancer is a prevalent malignancy affecting the urinary system, which presents a significant global health concern. Although there are many treatments for bladder cancer, identifying more effective drugs and methods remains an urgent problem. As a pivotal component of contemporary medical practice, traditional Chinese medicine (TCM) assumes a crucial role in the realm of anti-tumor therapy, especially with the identification of active ingredients and successful exploration of pharmacological effects. Febrifugine, identified as a quinazoline-type alkaloid compound extracted from the Cytidiaceae family plant Huangchangshan, exhibits heightened sensitivity to bladder cancer cells in comparison to control cells (non-cancer cells) group. The proliferation growth of bladder cancer cells T24 and SW780 was effectively inhibited by Febrifugine, and the IC50 was 0.02 and 0.018 µM respectively. Febrifugine inhibits cell proliferation by suppressing DNA synthesis and induces cell death by reducing steroidogenesis and promoting apoptosis. Combined with transcriptome analysis, Febrifugine was found to downregulate low density lipoprotein receptor-associated protein, lanosterol synthase, cholesterol biosynthesis second rate-limiting enzyme, 7-dehydrocholesterol reductase, flavin adenine dinucleotide dependent oxidoreductase and other factors to inhibit the production of intracellular steroids in bladder cancer T24 cells. The results of animal experiments showed that Febrifugine could inhibit tumor growth. In summary, the effect of Febrifugine on bladder cancer is mainly through reducing steroid production and apoptosis. Therefore, this study contributes to the elucidation of Febrifugine's potential as an inhibitor of bladder cancer and establishes a solid foundation for the future development of novel therapeutic agents targeting bladder cancer.

Synergistic anti-tumor effects of lenalidomide and gefitinib by upregulating ADRB2 and inactivating the mTOR/PI3K/AKT signaling pathway in lung adenocarcinoma.

Gefitinib is commonly used to be the first-line therapy for advanced non-small cell lung cancer (NSCLC). Therapeutic effect of gefitinib is reduced due to acquired resistance, and combined treatment is recommended. In this research, we planned to explore the impacts of combined treatment of lenalidomide and gefitinib on gefitinib-sensitive or -resistant NSCLC cells. The co-treatment results demonstrated that enhanced antitumor impact on NSCLC cell growth, migration, invasion, cell cycle process and apoptosis. The tumor-bearing mouse models were established using PC9/GR cells. In vivo assays also showed that lenalidomide and gefitinib synergistically inhibited mouse tumor growth along increased the survival of mice. ADRB2 was identified as a lowly expressed gene in PC9/GR cells and LUAD tumor tissues. LUAD patients with high ADRB2 expression were indicated with favorable survival outcomes. Moreover, ADRB2 was upregulated in lenalidomide and/or gefitinib-treated PC9/GR cells. ADRB2 deficiency partially offsets the suppressive impacts of lenalidomide and gefitinib co-treatment on the viability and proliferation of PC9/GR cells. Additionally, lenalidomide and gefitinib cotreatment significantly inactivated the mTOR/PI3K/AKT signaling pathway compared with each treatment alone. Rescue assays were performed to explore whether lenalidomide and gefitinib synergistically inhibited the growth of PC9/GR cells via the PI3K/AKT pathway. PI3K activator SC79 significantly restored reduced cell proliferation, migration and invasion along with elevated cell cycle arrest and apoptosis caused by lenalidomide and gefitinib cotreatment. In conclusion, lenalidomide and gefitinib synergistically suppressed LUAD progression and attenuated gefitinib resistance by upregulating ADRB2 and inactivating the mTOR/PI3K/AKT signaling pathway in lung adenocarcinoma.

Recent Advances in Structural Optimization of Quinazoline-Based Protein Kinase Inhibitors for Cancer Therapy (2021-Present).

Cancer is a complicated, multifaceted disease that can impact any organ in the body. Various chemotherapeutic agents have a low selectivity and are very toxic when used alone or in combination with others. Resistance is one of the most important hurdles that develop due to the use of many anticancer therapeutics. As a result, treating cancer requires a target-specific palliative care strategy. Remarkable scientific discoveries have shed light on several of the molecular mechanisms underlying cancer, resulting in the development of various targeted anticancer agents. One of the most important heterocyclic motifs is quinazoline, which has a wide range of biological uses and chemical reactivities. Newer, more sophisticated medications with quinazoline structures have been found in the last few years, and great strides have been made in creating effective protocols for building these pharmacologically active scaffolds. A new class of chemotherapeutic agents known as quinazoline-based derivatives possessing anticancer properties consists of several well-known compounds that block different protein kinases and other molecular targets. This review highlights recent updates (2021-2024) on various quinazoline-based derivatives acting against different protein kinases as anticancer chemotherapeutics. It also provides guidance for the design and synthesis of novel quinazoline analogues that could serve as lead compounds.

Leucine Zipper Downregulated in Cancer-1 Interacts with Clathrin Adaptors to Control Epidermal Growth Factor Receptor (EGFR) Internalization and Gefitinib Response in EGFR-Mutated Non-Small Cell Lung Cancer.

The epidermal growth factor receptor (EGFR) is a common driver of non-small cell lung cancer (NSCLC). Clathrin-mediated internalization (CMI) sustains EGFR signaling. AXL is associated with resistance to EGFR-tyrosine kinase inhibitors (TKIs) in EGFR-mutated (EGFRM) NSCLC. We investigated the effects of Leucine zipper downregulated in cancer-1 (LDOC1) on EGFR CMI and NSCLC treatment. Coimmunoprecipitation, double immunofluorescence staining, confocal microscopy analysis, cell surface labelling assays, and immunohistochemistry studies were conducted. We revealed that LDOC1 interacts with clathrin adaptors through binding motifs. LDOC1 depletion promotes internalization and plasma membrane recycling of EGFR in EGFRM NSCLC PC9 and HCC827 cells. Membranous and cytoplasmic EGFR decreased and increased, respectively, in LDOC1 (-) NSCLC tumors. LDOC1 depletion enhanced and sustained activation of EGFR, AXL, and HER2 and enhanced activation of HER3 in PC9 and HCC827 cells. Sensitivity to first-generation EGFR-TKIs (gefitinib and erlotinib) was significantly reduced in LDOC1-depleted PC9 and HCC827 cells. Moreover, LDOC1 downregulation was significantly associated (p < 0.001) with poor overall survival in patients with EGFRM NSCLC receiving gefitinib (n = 100). In conclusion, LDOC1 may regulate the efficacy of first-generation EGFR-TKIs by participating in the CMI of EGFR. Accordingly, LDOC1 may function as a prognostic biomarker for EGFRM NSCLC.

Phase I study of peposertib and avelumab with or without palliative radiotherapy in patients with advanced solid tumors.

INTRODUCTION: We report results from a phase I, three-part, dose-escalation study of peposertib, a DNA-dependent protein kinase inhibitor, in combination with avelumab, an immune checkpoint inhibitor, with or without radiotherapy in patients with advanced solid tumors. MATERIALS AND METHODS: Peposertib 100-400 mg twice daily (b.i.d.) or 100-250 mg once daily (q.d.) was administered in combination with avelumab 800 mg every 2 weeks in Part A or avelumab plus radiotherapy (3 Gy/fraction × 10 days) in Part B. Part FE assessed the effect of food on the pharmacokinetics of peposertib plus avelumab. The primary endpoint in Parts A and B was dose-limiting toxicity (DLT). Secondary endpoints were safety, best overall response per RECIST version 1.1, and pharmacokinetics. The recommended phase II dose (RP2D) and maximum tolerated dose (MTD) were determined in Parts A and B. RESULTS: In Part A, peposertib doses administered were 100 mg (n = 4), 200 mg (n = 11), 250 mg (n = 4), 300 mg (n = 6), and 400 mg (n = 4) b.i.d. Of DLT-evaluable patients, one each had DLT at the 250-mg and 300-mg dose levels and three had DLT at the 400-mg b.i.d. dose level. In Part B, peposertib doses administered were 100 mg (n = 3), 150 mg (n = 3), 200 mg (n = 4), and 250 mg (n = 9) q.d.; no DLT was reported in evaluable patients. Peposertib 200 mg b.i.d. plus avelumab and peposertib 250 mg q.d. plus avelumab and radiotherapy were declared as the RP2D/MTD. No objective responses were observed in Part A or B; one patient had a partial response in Part FE. Peposertib exposure was generally dose proportional. CONCLUSIONS: Peposertib doses up to 200 mg b.i.d. in combination with avelumab and up to 250 mg q.d. in combination with avelumab and radiotherapy were tolerable in patients with advanced solid tumors; however, antitumor activity was limited. GOV IDENTIFIER: NCT03724890.

Evodiamine inhibits EPRS expression to regulate glutamate metabolism and proliferation of oral squamous cell carcinoma cells.

The effects of evodiamine (EVO) on oral squamous cell carcinoma (OSCC) are not yet understood. Based on our earlier findings, we hypothesized that evodiamine may affect OSCC cell proliferation and glutamate metabolism by modulating the expression of EPRS (glutamyl-prolyl-tRNA synthetase 1). From GEPIA, we obtained EPRS expression data in patients with OSCC as well as survival prognosis data. An animal model using Cal27 cells in BALB/c nude mice was established. The expression of EPRS was assessed by immunofluorescence, Western blotting, and quantitative PCR. Glutamate measurements were performed to evaluate the impact of evodiamine on glutamate metabolism of Cal27 and SAS tumor cells. transient transfection techniques were used to knock down and modulate EPRS in these cells. EPRS is expressed at higher levels in OSCC than in normal tissues, and it predicts poor prognosis in patients. In a nude mouse xenograft model, evodiamine inhibited tumor growth and the expression of EPRS. Evodiamine impacted cell proliferation, glutamine metabolism, and EPRS expression on Cal27 and SAS cell lines. In EPRS knockdown cell lines, both cell proliferation and glutamine metabolism are suppressed. EPRS's overexpression partially restores evodiamine's inhibitory effects on cell proliferation and glutamine metabolism. This study provides crucial experimental evidence supporting the potential therapeutic application of evodiamine in treating OSCC. Evodiamine exhibits promising anti-tumor effects by targeting EPRS to regulate glutamate metabolism.

Use of Neoadjuvant Vandetanib in Aggressive Pediatric Medullary Thyroid Carcinoma.

Novel use of vandetanib in a child with aggressive MTC with prolonged response to treatment.

Design, synthesis and evaluation of quinazoline derivatives as Gαq/11 proteins inhibitors against uveal melanoma.

Uveal melanoma (UM) represents the predominant ocular malignancy among adults, exhibiting high malignancy and proclivity for liver metastasis. GNAQ and GNA11 encoding Gαq and Gα11 proteins are key genes to drive UM, making the selective inhibition of Gαq/11 proteins to be a potential therapeutic approach for combating UM. In this study, forty-six quinazoline derivatives were designed, synthesized, and assessed for their ability to inhibit Gαq/11 proteins and UM cells. Compound F33 emerged as the most favorable candidate, and displayed moderate inhibitory activity against Gαq/11 proteins (IC50 = 9.4 µM) and two UM cell lines MP41 (IC50 = 6.7 µM) and 92.1 (IC50 = 3.7 µM). Being a small molecule inhibitor of Gαq/11 proteins, F33 could effectively suppress the activation of downstream signaling pathways in a dose-dependent manner, and significantly inhibits UM in vitro.F33 represents a promising lead compound for developing therapeutics for UM by targeting Gαq/11 proteins.

Rare HER2 L796P missense mutation promotes the growth and oncogenic signaling in breast cancer cells.

PURPOSE: This research aimed to find potential HER2 mutations that would have an impact on breast cancer and investigate the underlying mechanism. EXPERIMENTAL DESIGN: This study first investigated 238 pairs of breast cancer and para-cancerous tissue samples from patients on the targeted next-generation sequencing (tNGS) platform. CCK-8 and clone formation assay were used to investigate whether the mutation exerts proliferative effects on breast cancer cells. In addition, mass spectrometry-based comparative proteomic and phosphoproteomic analyses of the mutation types and wild types of MCF-7 cell lines were carried out. RESULTS: Among the identified mutations, a new mutation HER2 L796P promoted the proliferation of breast cancer cells and had resistance to lapatinib using CCK-8 cell proliferation assay and clone formation assay. The bioinformatic analysis showed that RAS family proteins and ERK phosphorylated proteins significantly increased in the L796P mutant cells. The Gene Ontology (GO) analysis revealed that L796P mutation affected the function of breast cancer at the level of upstream genes in the MAPK and PI3K-AKT-TOR pathways. CONCLUSIONS AND CLINICAL RELEVANCE: This study demonstrated that a rare mutation HER2 L796P could be a potential therapeutic target for the clinical management of breast cancer.

Novel quinazoline-based dual EGFR/c-Met inhibitors overcoming drug resistance for the treatment of NSCLC: Design, synthesis and anti-tumor activity.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) have demonstrated the ability to impede tumor cell proliferation by suppressing EGFR expression. Nonetheless, patients undergoing treatment may acquire resistance, which may occur through an EGFR-dependent (such as T790M mutation) or an EGFR-independent (such as c-Met amplification) manner. Therefore, developing dual-target inhibitors might present a potential avenue for addressing treatment-acquired resistance in patients. Herein, we designed, synthesized, and screened several novel 4-phenoxyquinazoline derivatives, aiming to identify a potent dual EGFR/c-Met inhibitor for the treatment of NSCLC, among which H-22 emerged as the most promising candidate exhibiting significant antitumor properties. Moreover, we assessed the in vitro inhibitory effect of H-22 on EGFR kinase and c-Met kinase in five cancer cell lines. In addition, a series of functional experiments (cell cycle, apoptosis assays, in vitro/in vivo animal model, etc.) were conducted to further investigate the anti-tumor mechanisms of H-22. The present study revealed that H-22 exhibited strong antitumor activity both in vitro and in vivo. Interestingly, H-22 exhibited anti-proliferative activity (2.27-3.35 µM) similar to Afatinib against all five cancer cells, with inhibitory functions against EGFRWT, EGFRL858R/T790M, and c-Met kinases at a concentration of 64.8, 305.4 and 137.4 nM, respectively. Cell cycle analysis indicated that the antiproliferative activity of H-22 was associated with its ability to cause G2/M arrest. Furthermore, in vivo data showed that H-22 could inhibit tumor growth in our xenograft models and induce apoptosis. Collectively, our findings uncovered that H-22 is a novel dual EGFR and c-Met inhibitor and a prospective anti-tumor therapeutic drug.

Cediranib ameliorates portal hypertensive syndrome via inhibition of VEGFR-2 signaling in cirrhotic rats.

Portal hypertension (PHT) is a syndrome caused by systemic and portal hemodynamic disturbances with the progression of cirrhosis. However, the exact mechanisms regulating angiogenesis-related responses in PHT remain unclear. Cediranib is a potent inhibitor of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, exhibiting a greater affinity for VEGFR-2. Liver cirrhosis was induced by common bile duct ligation (BDL) in Sprague-Dawley rats. Sham-operated rats were controls. BDL and sham rats were randomly allocated to receive Cediranib or vehicle after BDL. On the 28th day, portal hypertension related parameters were surveyed. Cediranib treatment could significantly reduce the portal pressure (PP) in BDL rats, while it did not affect the mean arterial pressure (MAP) in sham groups and BDL groups. Cediranib treatment could significantly affect the stroke volume (SV), cardiac output (CO), cardiac index (CI), systemic vascular resistance (SVR), superior mesenteric artery (SMA) flow and SMA resistance in BDL groups and BDL with Cediranib groups. Cediranib treatment could improve the mesenteric vascular remodeling and contractility. Cediranib treatment significantly reduced mesenteric vascular density. And phospho-VEGFR-2 was significantly downregulated by Cediranib. On the other hand, phospho-endothelial Nitric Oxide Synthases (phospho-eNOS) expressions were upregulated. Cediranib not only improved splanchnic hemodynamics, extrahepatic vascular remodeling and vasodilation, but also alleviated intrahepatic fibrosis and collagen deposition significantly. Cediranib treatment could reduce intrahepatic angiogenesis between BDL-vehicle and BDL-Cediranib rats. In conclusion, Cediranib could improve extrahepatic hyperdynamic circulation by inhibiting extrahepatic angiogenesis through inhibition of the VEGFR-2 signaling pathway, portal collateral circulation formation, as well as eNOS-mediated vasodilatation and vascular remodeling, and at the same time, Cediranib improved intrahepatic fibrogenesis and angiogenesis, which together alleviate cirrhotic PHT syndrome.

Randomized phase II study of preoperative afatinib in untreated head and neck cancers: predictive and pharmacodynamic biomarkers of activity.

There is no strong and reliable predictive biomarker in head and neck squamous cell carcinoma (HNSCC) for EGFR inhibitors. We aimed to identify predictive and pharmacodynamic biomarkers of efficacy of afatinib, a pan-HER tyrosine kinase inhibitor, in a window-of-opportunity trial (NCT01415674). Multi-omics analyses were carried out on pre-treatment biopsy and surgical specimen for biological assessment of afatinib activity. Sixty-one treatment-naïve and operable HNSCC patients were randomised to afatinib 40 mg/day for 21-28 days versus no treatment. Afatinib produced a high rate of metabolic response. Responders had a higher expression of pERK1/2 (P = 0.02) and lower expressions of pHER4 (P = 0.03) and pRB1 (P = 0.002) in pre-treatment biopsy compared to non-responders. At the cellular level, responders displayed an enrichment of tumor-infiltrating B cells under afatinib (P = 0.02). At the molecular level, NF-kappa B signaling was over-represented among upregulated genes in non-responders (P < 0.001; FDR = 0.01). Although exploratory, phosphoproteomics-based biomarkers deserve further investigations as predictors of afatinib efficacy.