Pre-clinical characterization of Dacomitinib (PF-00299804), an irreversible pan-ErbB inhibitor, combined with ionizing radiation for head and neck squamous cell carcinoma.
ABSTRACT: Epidermal growth factor receptor (EGFR) is over-expressed in nearly all cases of squamous cell carcinoma of the head and neck (SCCHN), and is an important driver of disease progression. EGFR targeted therapies have demonstrated clinical benefit for SCCHN treatment. In this report, we investigated the pre-clinical efficacy of Dacomitinib (PF-00299804), an irreversible pan-ErbB inhibitor, both alone and in combination with ionizing radiation (IR), a primary curative modality for SCCHN. One normal oral epithelial (NOE) and three SCCHN (FaDu, UT-SCC-8, UT-SCC-42a) cell lines were used to conduct cell viability, clonogenic survival, cell cycle, and immunoblotting assays in vitro, using increasing doses of Dacomitinib (10-500 nM), both with and without IR (2-4 Gy). The FaDu xenograft model was utilized for tumor growth delay assays in vivo, and immunohistochemical analyses were conducted on extracted tumors. A dose-dependent reduction in cell viability and clonogenic survival after Dacomitinib treatment was observed in all three SCCHN models. Treatment led to a significant reduction in EGFR signalling, with a subsequent decrease in phosphorylation of downstream targets such as ERK, AKT, and mTOR. In vivo, Dacomitinib treatment delayed tumor growth, while decreasing phospho-EGFR and Ki-67 immunoexpression. These effects were further enhanced when combined with IR, both in vitro and in vivo. The preclinical data support the further evaluations of Dacomitinib combined with IR for the future management of patients with SCCHN.
Project description:In solid tumours millions of cells are shed into the blood circulation each day. Only a subset of these circulating tumour cells (CTCs) survive, many of them presumable because of their potential to form multi-cellular clusters also named spheroids. Tumour cells within these spheroids are protected from anoikis, which allows them to metastasize to distant organs or re-seed at the primary site. We used spheroid cultures of head and neck squamous cell carcinoma (HNSCC) cell lines as a model for such CTC clusters for determining the role of the epidermal growth factor receptor (EGFR) in cluster formation ability and cell survival after detachment from the extra-cellular matrix. The HNSCC cell lines FaDu, SCC-9 and UT-SCC-9 (UT-SCC-9P) as well as its cetuximab (CTX)-resistant sub-clone (UT-SCC-9R) were forced to grow in an anchorage-independent manner by coating culture dishes with the anti-adhesive polymer poly-2-hydroxyethylmethacrylate (poly-HEMA). The extent of apoptosis, clonogenic survival and EGFR signalling under such culture conditions was evaluated. The potential of spheroid formation in suspension culture was found to be positively correlated with the proliferation rate of HNSCC cell lines as well as their basal EGFR expression levels. CTX and gefitinib blocked, whereas the addition of EGFR ligands promoted anchorage-independent cell survival and spheroid formation. Increased spheroid formation and growth were associated with persistent activation of EGFR and its downstream signalling component (MAPK/ERK). Importantly, HNSCC cells derived from spheroid cultures retained their clonogenic potential in the absence of cell-matrix contact. Addition of CTX under these conditions strongly inhibited colony formation in CTX-sensitive cell lines but not their resistant subclones. Altogether, EGFR activation was identified as crucial factor for anchorage-independent survival of HNSCC cells. Targeting EGFR in CTC cluster formation might represent an attractive anti-metastatic treatment approach in HNSCC.
Project description:A novel pan ERBB inhibitor PF-00299804 (dacomitinib) is currently in phase II clinical trials in glioblastoma multiforme (GBM) patients; however its pre-clinical efficacy in GBMs has not been tested. In this study, we evaluated the efficacy of dacomitinib alone or in combination with PI3K/mTOR dual inhibitor PF-05212384 in GBM and assessed the mechanisms of resistance and the molecular determinants of response. A panel of established and patient derived primary GBM lines that present different molecular profiles and also the GBM lines engineered to express EGFRvIII mutant or PTEN were treated with either dacomitinib, PF-05212384, or combination and assessed for their viability and changes in EGFR/PI3K/mTOR signaling. We show that dacomitinib significantly reduced phosphorylated EGFR in all the GBM lines but did not show a dose-dependent response on cell viability in a majority of the lines tested. Multiple lesions in the receptor tyrosine kinases (RTKs) pathway including PTEN mutation, co-activation of RTKs, and EGFRvIII mutation resulted in unaltered active status of PI3K/mTOR in the GBM lines even in the presence of EGFR inhibition. Blocking PI3K/mTOR dramatically inhibited cell proliferation in most GBM lines and enhanced dacomitinib induction of apoptosis in a GBM line that has both EGFR amplification and EGFR-independent PI3K activation. These data suggest molecular profiling of EGFR/PI3K/PTEN status to select GBM patients for EGFR or/and PI3K/mTOR targeted therapies.
Project description:<h4>Purpose</h4>This randomized, open-label trial compared dacomitinib (PF-00299804), an irreversible inhibitor of human epidermal growth factor receptors (EGFR)/HER1, HER2, and HER4, with erlotinib, a reversible EGFR inhibitor, in patients with advanced non-small-cell lung cancer (NSCLC).<h4>Patients and methods</h4>Patients with NSCLC, Eastern Cooperative Oncology Group performance status 0 to 2, no prior HER-directed therapy, and one/two prior chemotherapy regimens received dacomitinib 45 mg or erlotinib 150 mg once daily.<h4>Results</h4>One hundred eighty-eight patients were randomly assigned. Treatment arms were balanced for most clinical and molecular characteristics. Median progression-free survival (PFS; primary end point) was 2.86 months for patients treated with dacomitinib and 1.91 months for patients treated with erlotinib (hazard ratio [HR] = 0.66; 95% CI, 0.47 to 0.91; two-sided P = .012); in patients with KRAS wild-type tumors, median PFS was 3.71 months for patients treated with dacomitinib and 1.91 months for patients treated with erlotinib (HR = 0.55; 95% CI, 0.35 to 0.85; two-sided P = .006); and in patients with KRAS wild-type/EGFR wild-type tumors, median PFS was 2.21 months for patients treated with dacomitinib and 1.84 months for patients treated with erlotinib (HR = 0.61; 95% CI, 0.37 to 0.99; two-sided P = .043). Median overall survival was 9.53 months for patients treated with dacomitinib and 7.44 months for patients treated with erlotinib (HR = 0.80; 95% CI, 0.56 to 1.13; two-sided P = .205). Adverse event-related discontinuations were uncommon in both arms. Common treatment-related adverse events were dermatologic and gastrointestinal, predominantly grade 1 to 2, and more frequent with dacomitinib.<h4>Conclusion</h4>Dacomitinib demonstrated significantly improved PFS versus erlotinib, with acceptable toxicity. PFS benefit was observed in most clinical and molecular subsets, notably KRAS wild-type/EGFR any status, KRAS wild-type/EGFR wild-type, and EGFR mutants.
Project description:The promising treatment combination of ionizing radiation (IR) with a hypoxia-activated prodrug (HAP) is based on biological cooperation. Here we investigated the hypoxia-activated prodrug evofosfamide in combination with different treatment regimens of IR against lung A549- and head&neck UT-SCC-14-derived tumor xenografts. DNA damage-related endpoints and clonogenic cell survival of A549 and UT-SCC-14 carcinoma cells were probed under normoxia and hypoxia.Evofosfamide (TH-302) induced DNA-damage and a dose-dependent antiproliferative response in A549 cells on cellular pretreatment under hypoxia, and supra-additively reduced clonogenic survival in combination with IR. Concomitant treatment of A549-derived tumor xenografts with evofosfamide and fractionated irradiation induced the strongest treatment response in comparison to the corresponding neoadjuvant and adjuvant regimens. Adjuvant evofosfamide was more potent than concomitant and neoadjuvant evofosfamide when combined with a single high dose of IR. Hypoxic UT-SCC-14 cells and tumor xenografts thereof were resistant to evofosfamide alone and in combination with IR, most probably due to reduced P450 oxidoreductase expression, which might act as major predictive determinant of sensitivity to HAPs.In conclusion, evofosfamide with IR is a potent combined treatment modality against hypoxic tumors. However, the efficacy and the therapeutic outcome of this combined treatment modality is, as indicated here in preclinical tumor models, dependent on scheduling parameters and tumor type, which is most probably related to the status of respective HAP-activating oxidoreductases. Further biomarker development is necessary for the launch of successful clinical trials.
Project description:Uterine serous carcinoma (USC) is an aggressive subtype of endometrial cancer that carries an extremely poor prognosis. Up to 35 % of USC may overexpress the epidermal growth factor receptor-2 (HER2/neu) at strong (i.e., 3+) level by immunohistochemistry (IHC) or harbor HER2/neu gene amplification by fluorescence in situ hybridization (FISH). In this study, we assessed the sensitivity of a panel of USC cell lines with and without HER2/neu gene amplification to dacomitinib (PF-00299804), an irreversible pan-human epidermal growth factor receptor tyrosine kinase inhibitor. Eight primary cell lines (i.e., four harboring HER2/neu gene amplification by FISH and four FISH- cell lines), all demonstrating similar in vitro growth rates, were evaluated in viability/proliferation assays. The effect of dacomitinib on cell growth, cell cycle distribution, and signaling was determined using flow cytometry-based assays. Dacomitinib caused a significantly stronger growth inhibition in HER2/neu FISH+ USC cell lines when compared to FISH- USC (dacomitinib half maximal inhibitory concentration (IC50) mean ± SEM = 0.02803 ± 0.003355 ?M in FISH+ versus 1.498 ± 0.2209 ?M in FISH- tumors, P < 0.0001). Dacomitinib growth inhibition was associated with a significant and dose-dependent decline in phosphorylated HER2/neu and S6 transcription factor and a dose-dependent and time-dependent cell cycle arrest in G0/G1 in FISH+ USC. Dacomitinib is remarkably effective against chemotherapy-resistant HER2/neu gene-amplified USC. Clinical studies with dacomitinib in HER2/neu FISH+ USC patients resistant to standard salvage chemotherapy are warranted.
Project description:This phase 2 trial (ClinicalTrials.gov identifier NCT00548093) assessed the efficacy, safety, and impact on health-related quality of life of dacomitinib (PF-00299804), an irreversible tyrosine kinase inhibitor (TKI) of human epidermal growth factor receptors (EGFR)/HER1, HER2, and HER4, in patients with KRAS wild-type non-small cell lung cancer (NSCLC).Patients with advanced NSCLC, progression on 1 or 2 regimens of chemotherapy and erlotinib, KRAS wild-type or known EGFR-sensitizing mutant tumor, and Eastern Cooperative Oncology Group performance status of 0 to 2 received 45 mg of dacomitinib once daily continuously in 21-day cycles.A total of 66 patients enrolled (adenocarcinoma, n = 50; those without adenocarcinoma [nonadenocarcinoma], n = 16). The objective response rate (ORR) for patients with adenocarcinoma (primary endpoint) was 5% (2 partial responses; 1-sided P = .372 for null hypothesis [H0 ]: ORR ? 5%) and 6% (1 partial response) for patients with nonadenocarcinoma. Responders included: 2 of 25 EGFR mutation-positive tumors; 1 of 3 EGFR wild-type with HER2 amplification. Median progression-free survival was 12 weeks overall (n = 66) and 18 weeks (n = 26) for patients with EGFR mutation-positive tumors. Common treatment-related adverse events were of grade 1 or 2 severity, manageable with standard supportive care, and included diarrhea (grade 3 [G3], 12%), acneiform dermatitis (G3, 6%), exfoliative rash (G3, 3%), dry skin (G3, 0%), fatigue (G3, 3%), and stomatitis (G3, 2%). Six patients (9%) discontinued due to treatment-related adverse events. By patient report, NSCLC symptoms of dyspnea, cough, and pain (chest, arm/shoulder) showed improvement first observed after 3 weeks on therapy.Dacomitinib demonstrated preliminary activity and acceptable tolerability in heavily pretreated patients, and may offer benefit in molecularly defined patient subsets.
Project description:<h4>Background</h4>A major objective in the management of human papillomavirus (HPV)-positive squamous cell carcinoma of the head and neck (SCCHN) is to reduce long-term functional ramifications while maintaining oncological outcomes. This study examined the metabolic profile of HPV-positive SCCHN and the potential role of anti-metabolic therapeutics to achieve radiosensitisation as a potential means to de-escalate radiation therapy.<h4>Methods</h4>Three established HPV-positive SCCHN cell lines were studied (UM-SCC-104, UPCI:SCC154, and VU-SCC-147), together with a typical <i>TP53</i> mutant HPV-negative SCCHN cell line (UM-SCC-81B) for comparison. Metabolic profiling was performed using extracellular flux analysis during specifically designed mitochondrial and glycolytic stress tests. Sensitivity to ionising radiation (IR) was evaluated using clonogenic assays following no treatment, or treatment with: 25 mM 2-deoxy-D-glucose (glycolytic inhibitor) alone; 20 mM metformin (electron transport chain inhibitor) alone; or 25 mM 2-deoxy-D-glucose and 20 mM metformin combined. Expression levels of p53 and reporters of p53 function (MDM2, p53, Phospho-p53 [Ser15], TIGAR and p21 [CDKN1A]) were examined by western blotting.<h4>Results</h4>HPV-positive SCCHN cell lines exhibited a diverse metabolic phenotype, displaying robust mitochondrial and glycolytic reserve capacities. This metabolic profile, in turn, correlated with IR response following administration of anti-metabolic agents, in that both 2-deoxy-D-glucose and metformin were required to significantly potentiate the effects of IR in these cell lines.<h4>Conclusions</h4>In contrast to our recently published data on HPV-negative SCCHN cells, which display relative glycolytic dependence, HPV-positive SCCHN cells can only be sensitised to IR using a complex anti-metabolic approach targeting both mitochondrial respiration and glycolysis, reflecting their metabolically diverse phenotype. Notionally, this may provide an attractive platform for treatment de-intensification in the clinical setting by facilitating IR dose reduction to minimise the impact of treatment on long-term function.
Project description:Despite recent advances in radiotherapy and chemotherapy, survival rates for squamous cell carcinoma of the head and neck (SCCHN) have remained poor. The focus of SCCHN therapy has more recently shifted to the molecular level, particularly the epidermal growth factor receptor (EGFR/ErbB) pathway. Several agents that target the EGFR pathway, including monoclonal antibodies and tyrosine kinase inhibitors, are under investigation for SCCHN. Searches of PubMed and results of key oncology congresses were performed to identify relevant articles and abstracts. The EGFR-targeted monoclonal antibody cetuximab is approved for the treatment of locally advanced SCCHN in combination with radiotherapy, for first-line treatment of recurrent or metastatic SCCHN in combination with platinum-based chemotherapy and 5-fluorouracil, and for recurrent or metastatic SCCHN following progression with platinum-based chemotherapy. Other investigational EGFR-targeted monoclonal antibodies (e.g., panitumumab, nimotuzumab, zalutumumab) are in clinical development for SCCHN. Inhibition of the tyrosine kinase domain of EGFR has also been explored as a therapeutic approach in SCCHN using small-molecule reversible inhibitors, such as gefitinib and erlotinib. However, a key challenge in SCCHN is the development of resistance, and strategies are being pursued to delay or overcome resistance to EGFR-targeted agents. These strategies include development of agents that inhibit multiple ErbB receptors simultaneously (e.g., lapatinib) or that bind multiple ErbB family receptors irreversibly (e.g., afatinib, PF-00299804) and investigation of combinations of agents that target multiple pathways implicated in the pathogenesis of SCCHN. Ongoing large clinical trials are evaluating these emerging agents and combinations for the treatment of SCCHN.
Project description:<h4>Background</h4>Dacomitinib (PF-00299804) is an oral, irreversible, small molecule inhibitor of human epidermal growth factor receptor-1, -2, and -4 tyrosine kinases.<h4>Methods</h4>This phase I, open-label, dose-escalation study (clinicaltrials.gov: NCT00783328) primarily evaluated the safety and tolerability of dacomitinib by dose-limiting toxicity (DLT), and determined the clinically recommended phase II dose (RP2D) in Japanese patients with advanced solid tumors. Dacomitinib was administered orally at three dose levels (15, 30, or 45 mg once daily [QD]). Patients initially received a single dose, and after 9 days of follow-up, continuously QD in 21-day cycles. Endpoints included pharmacokinetics (PK) and antitumor activity.<h4>Results</h4>Thirteen patients were assigned to the three dose levels (15 mg cohort: n?=?3; 30 mg cohort: n?=?3; 45 mg cohort: n?=?7) according to a traditional '3?+?3' design. None of the treated patients experienced a DLT. Toxicities were manageable and similar in type to those observed in other studies. PK concentration parameters increased with dose over the range evaluated, with no evidence of accumulation over time. Of 13 evaluable patients, one with NSCLC (adenocarcinoma) had a partial response and nine patients had stable disease.<h4>Conclusions</h4>Dacomitinib 45 mg QD was defined as the RP2D and demonstrated preliminary activity in Japanese patients with advanced solid tumors.
Project description:Aberrant epidermal growth factor (EGF) signaling is associated with tumor growth in squamous cell carcinoma of the head and neck in humans (HNSCC), and is a major focus of targeted therapy. Cetuximab, a monoclonal antibody against EGFR, has been successful at prolonging survival but has only a 10% tumor shrinkage response rate in a clinical setting. The goal of this study was to compare dacomitinib (PF-00299804), a next generation small molecule tyrosine kinase inhibitor that irreversibly blocks multiple HER family receptors (HER-1 (EGFR), HER-2 and HER-4 tyrosine kinases), to cetuximab, the current FDA approved anti-EGFR medication for HNSCC and erlotinib, an EGFR specific small molecule tyrosine kinase inhibitor. Dacomitinib, erlotinib and cetuximab were tested in a panel of 27 HNSCC cell lines. Treatment with 100 ug/ml of cetuximab or 1 uM of erlotinib inhibited growth by at least 50% in 7/27 cell lines, while treatment with 1 uM of dacomitinib had similar growth inhibition in 17/27 lines. Cell lines representing three levels of sensitivity to dacomitinib were further examined using Western blots, cell cycle and apoptosis analysis. Treatment with 100 nM of dacomitinib reduced EGFR activity and downstream AKT and ERK pathways more effectively than treatment with 100 ug/ml of cetuximab in all ten tested lines. Although both compounds induced apoptosis at similar levels, dacomitinib caused greater G0/G1 arrest. Sensitivity to EGFR blockade was associated with levels of EGFR and ERK and was not associated with common oncogenic mutations and copy number variations. Phosphorylated and total EGFR and ERK levels correlate with sensitivity to both cetuximab and dacomitinib. Three of the four lines in the exquisitely sensitive group had the highest levels of phosphorylated and total EGFR and ERK among the ten lines selected, while the three resistant lines collectively had the lowest levels. Neither pAKT nor tAKT was associated with sensitivity.