Cetuximab Resistance Research Articles

Decreased SMAD4 expression is associated with induction of epithelial-to-mesenchymal transition and cetuximab resistance in head and neck squamous cell carcinoma

Epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and cetuximab, a monoclonal antibody targeting this receptor, is widely used to treat these patients. In the following investigation, we examined the role of SMAD4 down-regulation in mediating epithelial-to-mesenchymal transition (EMT) and cetuximab resistance in HNSCC. We determined that SMAD4 downregulation was significantly associated with increased cell motility, increased expression of vimentin, and cetuximab resistance in HNSCC cell lines. In the HNSCC genomic dataset obtained from The Cancer Genome Atlas, SMAD4 was altered in 20/279 (7%) of HNSCC via homozygous deletion, and nonsense, missense, and silent mutations. When SMAD4 expression was compared with respect to human papillomavirus (HPV) status, HPV-positive tumors had higher expression compared to HPV-negative tumors. Furthermore, higher SMAD4 expression also correlated with higher CDKN2A (p16) expression. Our data suggest that SMAD4 down-regulation plays an important role in the induction of EMT and cetuximab resistance. Patients with higher SMAD4 expression may benefit from cetuximab use in the clinic.

AKT1 E17K in Colorectal Carcinoma Is Associated with BRAF V600E but Not MSI-H Status: A Clinicopathologic Comparison to PIK3CA Helical and Kinase Domain Mutants.

The PI3K/AKT/mTOR pathway is activated through multiple mechanisms in colorectal carcinoma. Here, the clinicopathologic and molecular features of AKT1 E17K-mutated colorectal carcinoma in comparison with PIK3CA-mutated colorectal carcinoma are described in detail. Interestingly, in comparison with PIK3CA mutants, AKT1 E17K was significantly associated with mucinous morphology and concurrent BRAF V600E mutation. Among PIK3CA mutants, exon 21 mutations were significantly associated with BRAF V600E mutation, MSI-H status, and poor differentiation, while exon 10 mutations were associated with KRAS/NRAS mutations. Three of four AKT1 mutants with data from both primary and metastatic lesions had concordant AKT1 mutation status in both. Both AKT1- and PIK3CA-mutant colorectal carcinoma demonstrated frequent loss of PTEN expression (38% and 34%, respectively) and similar rates of p-PRAS 40 expression (63% and 50%, respectively). Both patients with AKT1 E17K alone had primary resistance to cetuximab, whereas 7 of 8 patients with PIK3CA mutation alone experienced tumor shrinkage or stability with anti-EGFR therapy. These results demonstrate that AKT1 E17K mutation in advanced colorectal carcinoma is associated with mucinous morphology, PIK3CA wild-type status, and concurrent RAS/RAF mutations with similar pattern to PIK3CA exon 21 mutants. Thus, AKT1 E17K mutations contribute to primary resistance to cetuximab and serve as an actionable alteration. This first systematic study of AKT1 and PIK3CA hotspot mutations and their association with cetuximab resistance and BRAF V600E mutation has important ramifications for the development of personalized medicine, particularly in identifying patient candidates for PI3K or AKT inhibitors.

Safety and Activity of the First-in-Class Sym004 Anti-EGFR Antibody Mixture in Patients with Refractory Colorectal Cancer

Tumor growth in the context of EGFR inhibitor resistance may remain EGFR-dependent and is mediated by mechanisms including compensatory ligand upregulation and de novo gene alterations. Sym004 is a two-antibody mixture targeting nonoverlapping EGFR epitopes. In preclinical models, Sym004 causes significant EGFR internalization and degradation, which translates into superior growth inhibition in the presence of ligands. In this phase I trial, we observed grade 3 skin toxicity and hypomagnesemia as mechanism-based dose-limiting events during dose escalation. In dose-expansion cohorts of 9 and 12 mg/kg of Sym004 weekly, patients with metastatic colorectal cancer and acquired EGFR inhibitor resistance were enrolled; 17 of 39 patients (44%) had tumor shrinkage, with 5 patients (13%) achieving partial response. Pharmacodynamic studies confirmed marked Sym004-induced EGFR downmodulation. MET gene amplification emerged in 1 patient during Sym004 treatment, and a partial response was seen in a patient with EGFR(S492R) mutation that is predictive of cetuximab resistance. Potent EGFR downmodulation with Sym004 in patients with metastatic colorectal cancer and acquired resistance to cetuximab/panitumumab translates into significant antitumor activity and validates the preclinical hypothesis that a proportion of tumors remains dependent on EGFR signaling. Further clinical development and expanded correlative analyses of response patterns with secondary RAS/EGFR mutations are warranted.

CCI-779 (Temsirolimus) exhibits increased anti-tumor activity in low EGFR expressing HNSCC cell lines and is effective in cells with acquired resistance to cisplatin or cetuximab.

BackgroundThe mammalian target of rapamycin (mTOR) signaling pathway plays a pivotal role in numerous cellular processes involving growth, proliferation and survival. The purpose of this study was to investigate the anti-tumoral effect of the mTOR inhibitor (mTORi) CCI-779 in HNSCC cell lines and its potency in cisplatin- and cetuximab-resistant cells.MethodsA panel of 10 HNSCC cell lines with differences in TP53 mutational status and basal cisplatin sensitivity and two isogenic models of acquired resistance to cisplatin and cetuximab, respectively were studied. Cell survival after treatment with CCI-779, cisplatin and cetuximab alone or in combination was determined by MTT assays. Potential predictive biomarkers for tumor cell sensitivity to CCI-779 were evaluated.ResultsWe observed considerable heterogeneity in sensitivity of HNSCC cell lines to CCI-779 monotherapy. Sensitivity was observed in TP53 mutated as well as wild-type cell lines. Total and p-EGFR expression levels but not the basal activity of the mTOR and MAPK signaling pathways were correlated with sensitivity to CCI-779. Resistant cells with increased EGFR activation could be sensitized by the combination of CCI-779 with cetuximab. Interestingly, cell lines with acquired resistance to cisplatin displayed a higher sensitivity to CCI-779 whereas cetuximab-resistant cells were less sensitive to the drug, but could be sensitized to CCI-779 by EGFR blockade.ConclusionsActivity of CCI-779 in HNSCC cells harboring TP53 mutations and displaying a phenotype of cisplatin resistance suggests its clinical potential even in patients with dismal outcome after current standard treatment. Cetuximab/mTORi combinations might be useful for treatment of tumors with high expression of EGFR/p-EGFR and/or acquired cetuximab resistance. This combinatorial treatment modality needs further evaluation in future translational and clinical studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0456-6) contains supplementary material, which is available to authorized users.

Abstract B39: Generation of head and neck cancer patient-derived xenografts with in vivo acquired cetuximab resistance

Abstract Background: Although cetuximab has demonstrated only modest response rates in head and neck squamous cell carcinoma (HNSCC) patients in clinical studies, most HNSCC cell line xenograft models have demonstrated sensitivity to cetuximab. We sought to characterize molecular and phenotypic changes associated with the acquisition of cetuximab resistance by evaluating patient-derived xenograft (PDX) models with and without cetuximab selection in vivo. Methods: A single PDX was established from a patient tongue HNSCC and a separate PDX from an independent patient lymph node by subcutaneous propagation in nod scid gamma (NSG) immunodeficient mice. Established PDXs were implanted into 2 flanks of NSG mice, and mice were either untreated (parental) or treated with cetuximab using modulated dosing dependent upon tumor growth (0.04-0.2 mg 1-2 times weekly) with the goal of PDX growth at the maximum dose. Tumor histology and short tandem repeat (STR) profiling were evaluated for HNSCC tumors and paired PDX models. Cell lines were derived from PDX tumors grown in NSG hypoxanthine-guanine phosphoribosyltransferase (Hprt)-deficient mice, allowing negative selection of mouse fibroblasts in vitro. Cell lines were retrovirally transduced to express either green fluorescent protein (GFP) or telomerase (HTERT). Cell lines were evaluated for presence of human cells using a PCR-based assay to detect human amelogenin alleles (sex id assay). Candidate gene mutations and protein/phosphoprotein levels will be evaluated in parental and cetuximab-selected PDXs using Ion Torrent and immunoblotting technologies, respectively. Results: Patient HNSCC tumors and paired parental PDXs displayed similar histologies and consistent STR profiles. Four of 14 PDXs derived from the tongue HNSCC displayed growth in the presence of cetuximab at the maximum dose, and of 18 PDXs derived from lymph node, 3 grew in the presence of cetuximab at the maximum dose. Average tumor volume at treatment initiation was 134 mm3 and average duration of treatment was 8 months. For tumors that eventually grew in presence of maximum dose cetuximab, initial tumor volume and duration of treatment were 156.5 mm3 and 9 months, respectively. Cetuximab-resistant tumors were heterogeneous regarding their resistance to cetuximab in a subsequent passage. Of the two cetuximab-resistant tumors STR evaluated, 1 was consistent with the patient and parental PDX and 1 could not be genotyped. In vitro cell cultures were established from parental PDX tumor cells transduced with HTERT and GFP, and these cells were confirmed to be of human origin by the sex id assay. Molecular profiling and establishment of cell lines derived from cetuximab-resistant, genotype confirmed PDXs are currently ongoing. Conclusions: It was possible to obtain PDX tumors grown under conditions of chronic treatment with cetuximab. However, dose modulation was required and propagation of the trait of cetuximab-resistant growth upon PDX passage was not guaranteed. The acquisition of cetuximab-resistant PDX tumors was a lengthy process that required care regarding dosing modulation and genotype validation. Once established, these models will provide valuable insights regarding molecular and phenotypic changes associated with acquired cetuximab resistance. Citation Format: Pei Zhou, Hua Li, Sarah Wheeler, Jennifer R. Grandis, Laura A. Stabile, Ann Marie Egloff. Generation of head and neck cancer patient-derived xenografts with in vivo acquired cetuximab resistance. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B39.

Abstract B43: HER2-amplified gastric and colorectal cancers: Mechanistic insights into HER2 targeting approaches

Abstract Only 10% of genetically unselected patients with chemorefractory metastatic colorectal cancer (mCRC) receive clinical benefit from therapy with the anti-EGFR antibodies cetuximab and panitumumab. Among non-responsive patients, 70% bear tumors harboring at least one alteration in KRAS, NRAS, BRAF and PIK3CA genes; therefore, the remaining 30% of “quadruple negative” cases display still-unidentified features that sustain primary resistance to EGFR-targeted therapies. By performing genotype-response correlations in a preclinical platform of patient-derived mCRC xenografts (xenopatients), our group has recently identified HER2 amplification as a new biomarker of resistance to cetuximab within a quadruple negative population. We found that the anti-HER2 monoclonal antibody trastuzumab, despite its demonstrated efficacy in patients with HER2-positive breast and gastric cancers, was completely ineffective in HER2-amplified mCRC xenopatients. Importantly, while the dual EGFR/HER2 small molecule inhibitor lapatinib induced disease stabilization, the combination of lapatinib with trastuzumab was synergistic, achieving rapid and long lasting tumor regressions. When the same regimens where applied to HER2-positive gastric cancer cells in vivo, trastuzumab and lapatinib as monotherapies induced disease stabilization, while the combination of both – similar to CRC – had stronger therapeutic effect. The aim of this study was to better characterize the molecular mechanisms underlying the synergistic effect of the combination therapy in HER2-positive gastric and colorectal cancers. We first generated three CRC cell lines (NCI-H508, DiFi and HDC-142) stably transfected with the HER2 oncogene and provided proof-of-concept of the causative role of HER2 in installing cetuximab resistance in mCRC. Indeed, while parental controls proved to be extremely sensitive to cetuximab, HER2 overexpressors displayed overt resistance, both in viability assays in vitro and in xenografts experiments in vivo. Moreover, NCI-H508-HER2 cells in vivo were poorly responsive to trastuzumab, while lapatinib induced disease stabilization and the combination of both achieved the strongest therapeutic effect. Overall, these responses were very similar to those observed for HER2-positive mCRC xenopatients, attesting to the reliability of this artificial cellular model for further mechanistic studies. We next analyzed the activation status of EGFR, HER2 and HER3 upon treatment with trastuzumab, lapatinib and combo and explored the signaling consequences of receptor blockade. Dose-response curves revealed that trastuzumab did not affect activation of EGFR and HER2, but was able to reduce HER3 phosphorylation; at variance, lapatinib and combo potently inhibited phosphorylation of all HERs in a similar manner, with no additive effect by combo. Interestingly, time-course experiments highlighted that treatment with lapatinib resulted in a paradoxical increase of HER3 expression and phosphorylation both in HER2-positive gastric cancer cells and in colorectal cancer cells. Importantly, adding trastuzumab to lapatinib completely neutralized lapatinib-induced HER3 hyper-phosphorylation, without affecting HER3 levels. Possibly, up-regulated HER3 could provide an alternate route to activate a “bypass track” signaling, attenuating the antitumor effects of lapatinib as monotherapy in these tumor settings. Full inhibition of a residual HER2 activity and consequent abolishment of HER3 phosphorylation achieved by the addition of trastuzumab could provide a mechanistic explanation of the synergic effect obtained with the combination therapy. Overall, these results provide new mechanistic insights into resistance and response to EGFR and HER2 targeted therapies and deserve further investigations to explore the key pathways involved in HER3 feedback up-regulation in the context of HER2-positive cancers. Citation Format: Simonetta Maria Leto, Francesco Sassi, Giorgia Migliardi, Eugenia Zanella, Irene Catalano, Andrea Bertotti, Livio Trusolino. HER2-amplified gastric and colorectal cancers: Mechanistic insights into HER2 targeting approaches. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B43.

Epidermal growth factor is a potential biomarker for poor cetuximab response in tongue cancer cells.

Head and neck squamous cell carcinoma is frequently associated with aberrant epidermal growth factor receptor (EGFR) signaling, which contributes to tumor growth. Here, the functional importance of EGFR ligands in relation to proliferation and sensitivity to the EGFR-targeted therapy cetuximab was investigated in three tongue cancer cell lines. The influence of epidermal growth factor (EGF), amphiregulin (AR), and epiregulin (EPR) on tumor cell proliferation and cetuximab response was evaluated by the addition of recombinant human (rh) proteins or by siRNA-mediated downregulation of the endogenous ligand production. The expression, activation and cellular distribution of EGFR were assessed by Western blot analysis and immunocytochemistry. EGF downregulation suppressed the proliferation of all investigated tumor cell lines, whereas the response to an increased level of EGF differed between EGFR-overexpressing and EGFR-non-overexpressing cell lines. Furthermore, tumor cells consistently displayed increased cetuximab resistance upon the addition of rhEGF, whereas EGF silencing was associated with an improved cetuximab response. The data regarding AR and EPR were inconclusive. Our data suggest that the amount of EGF is a determinant of the tumor cell proliferation rate and the response to cetuximab treatment in tongue cancer. Thus, EGF is a potential predictive biomarker of poor cetuximab response and a possible treatment target.

SP-050: Promising targets beyond EGFR

HNC represent a heterogeneous disease with regard to primary site, histology, molecular signature carcinogenesis and prognosis. EGFR represent a critical target for treatment approach both in tobacco/alcohol and HPV-induced HNC. A monoclonal antibody against EGFR (Cetuximab) has gained EMA and FDA approval to treat locally advanced HNC in combination with RT and in recurrent/metastatic setting in combination with platinum based CT. However cure rate and survival remain dismal. Therefore further drugs targeting molecular pathways associated with HNC are under evaluation. The specific targeting of new pathways (such as PI3K-AKT-mTOR pathway; JAK-STAT pathway; tumor suppressive miRNAs; Notch1 and 2; epithelial to mesenchymal transformation and immunitary check points) is needed to improve survival outcomes in HNSCC. Moreover second and third generation EGFR inhibitors have the ability to overcome Cetuximab resistance for example targeting the tumor-specific EGFR deletion mutant EGFR vIII. A promising approach is also to combine several target therapy to abrogate the cross-talk between various receptor TK cascades. Phase III are currently on going using combinations of PI3K inhibitors or mTOR inhibitors and chemotherapy in platinum resistant R/M HNC (6-8% of HNCs present PIK3CA mutations). Recently studies on JAK inhibitors showed their antitumor activity in combination with EGFR inhibitors. An emerging field of treatment is to act on immune system. HNC elicits T cell anergy in both peripheral and tumor infiltrating lymphocytes (TILs) In the tumor microenviroment a low level of antimuor cytokines and co-stimulatory mediators has been reported. On the other hand co-inhibitor signals are increased (cytotoxic T lymphocyte associated antigen 4 (CTLA4) and programmed death 1 (PD-1) Successful response depends on prevalence of co-stimulator and/or block of co-inhibitory signaling on immune system A new therapeutic paradigm is to use mAb against coinhibitory receptors CTLA4 and PD1. In particular objective responses with mAbs against PDL1 correlates with PDL! expression on tumour. Moreover as in the melanoma setting the use of antiPD1 AB (BMS936559) there is also growing enthusiasm for therapeutical vaccines against HPV16-18 and p53 respectively in HPV pos and neg disease

HER2 as a therapeutic target in head and neck squamous cell carcinoma.

The majority of patients with head and neck squamous cell carcinoma (HNSCC) present with advanced-stage disease. The current standard of care is surgery followed by adjuvant radiotherapy with or without chemotherapy or chemoradiation alone. The addition of cetuximab for the treatment of patients with locally advanced or recurrent/metastatic HNSCC has improved overall survival and locoregional control; however, responses are often modest, and treatment resistance is common. A variety of therapeutic strategies are being explored to overcome cetuximab resistance by blocking candidate proteins implicated in resistance mechanisms such as HER2. Several HER2 inhibitors are in clinical development for HNSCC, and HER2-targeted therapy has been approved for several cancers. This review focuses on the biology of HER2, its role in cancer development, and the rationale for clinical investigation of HER2 targeting in HNSCC.

Cetuximab-induced insulin-like growth factor receptor I activation mediates cetuximab resistance in gastric cancer cells

Epidermal growth factor receptor (EGFR) and insulin‑like growth factor receptor‑I (IGF‑IR) are frequently overexpressed in gastric cancercells. However, thesecells are resistant to the anti‑EGFR monoclonal antibody cetuximab. The aim of the present study was to determine whether cetuximab resistance in gastric cancercells resulted from activation of the IGF‑IR signaling pathway by cetuximab. The results demonstrated that EGFR phosphorylation was markedly inhibited in gastric cancer cell lines (SGC7901 and MGC803) which possessed functional K‑ras and BRAF following treatment with cetuximab. However, cetuximab treatment did not diminish cell viability; by contrast, IGF‑IR activation was observed. Knockdown of IGF‑IR or the use of an IGF‑IR inhibitor were found to increase the sensitivity of gastric cancercells to cetuximab. Furthermore, cetuximab induced phosphorylation of the non‑receptor tyrosine kinase c‑steroid receptor co‑activator (Src). Treatment of gastric cancercells with a Src inhibitor was shown to significantly reduce cetuximab‑induced phosphorylation of IGF‑IR as well as Src, which resulted in enhanced sensitivity to cetuximab treatment. In conclusion, the results of the present study demonstrated that cetuximab‑induced IGF‑IR activation was involved in cetuximab resistance in gastric cancercells and that Src was an important mediator for IGF‑IR activation.

Significant association of oncogene YAP1 with poor prognosis and cetuximab resistance in colorectal cancer patients.

Activation of YAP1, a novel oncogene in the Hippo pathway, has been observed in many cancers, including colorectal cancer. We investigated whether activation of YAP1 is significantly associated with prognosis or treatment outcomes in colorectal cancer. A gene expression signature reflecting YAP1 activation was identified in colorectal cancer cells, and patients with colorectal cancer were stratified into two groups according to this signature: activated YAP1 colorectal cancer (AYCC) or inactivated YAP1 colorectal cancer (IYCC). Stratified patients in five test cohorts were evaluated to determine the effect of the signature on colorectal cancer prognosis and response to cetuximab treatment. The activated YAP1 signature was associated with poor prognosis for colorectal cancer in four independent patient cohorts with stage I-III disease (total n = 1,028). In a multivariate analysis, the impact of the YAP1 signature on disease-free survival was independent of other clinical variables [hazard ratio (HR), 1.63; 95% confidence interval (CI), 1.25-2.13; P < 0.001]. In patients with stage IV colorectal cancer and wild-type KRAS, IYCC patients had a better disease control rate and progression-free survival (PFS) after cetuximab monotherapy than did AYCC patients; however, in patients with KRAS mutations, PFS duration after cetuximab monotherapy was not different between IYCC and AYCC patients. In multivariate analysis, the effect of YAP1 activation on PFS was independent of KRAS mutation status and other clinical variables (HR, 1.82; 95% CI, 1.05-3.16; P = 0.03). Activation of YAP1 is highly associated with poor prognosis for colorectal cancer and may be useful in identifying patients with metastatic colorectal cancer resistant to cetuximab.

Concurrent Targeting of KRAS and AKT by MiR-4689 Is a Novel Treatment Against Mutant KRAS Colorectal Cancer.

KRAS mutations are a major cause of drug resistance to molecular-targeted therapies. Aberrant epidermal growth factor receptor (EGFR) signaling may cause dysregulation of microRNA (miRNA) and gene regulatory networks, which leads to cancer initiation and progression. To address the functional relevance of miRNAs in mutant KRAS cancers, we transfected exogenous KRASG12V into human embryonic kidney 293 and MRC5 cells with wild-type KRAS and BRAF genes, and we comprehensively profiled the dysregulated miRNAs. The result showed that mature miRNA oligonucleotide (miR)-4689, one of the significantly down-regulated miRNAs in KRASG12V overexpressed cells, was found to exhibit a potent growth-inhibitory and proapoptotic effect both in vitro and in vivo. miR-4689 expression was significantly down-regulated in cancer tissues compared to normal mucosa, and it was particularly decreased in mutant KRAS CRC tissues. miR-4689 directly targets v-ki-ras2 kirsten rat sarcoma viral oncogene homolog (KRAS) and v-akt murine thymoma viral oncogene homolog 1(AKT1), key components of two major branches in EGFR pathway, suggesting KRAS overdrives this signaling pathway through inhibition of miR-4689. Overall, this study provided additional evidence that mutant KRAS functions as a broad regulator of the EGFR signaling cascade by inhibiting miR-4689, which negatively regulates both RAS/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. These activities indicated that miR-4689 may be a promising therapeutic agent in mutant KRAS CRC.

The anti-HER3 antibody patritumab abrogates cetuximab resistance mediated by heregulin in colorectal cancer cells.

We previously showed that tumor-derived heregulin, a ligand for HER3, is associated with both de novo and acquired resistance to cetuximab. We have now examined whether patritumab, a novel neutralizing monoclonal antibody to HER3, is able to overcome such resistance. Human colorectal cancer (DiFi) cells that are highly sensitive to cetuximab were engineered to stably express heregulin by retroviral infection, and the effects of cetuximab and patritumab on the resulting DiFi-HRG cells were examined. DiFi-HRG cells released substantial amounts of heregulin and showed resistance to cetuximab. Cetuximab alone inhibited EGFR and ERK phosphorylation in DiFi-HRG cells, but it had no effect on the phosphorylation of HER2, HER3, or AKT, suggesting that sustained AKT activation by HER2 and HER3 underlies cetuximab resistance in these cells. In contrast, patritumab in combination with cetuximab markedly inhibited the phosphorylation of EGFR, HER2, HER3, ERK, and AKT. The combination therapy also inhibited the growth of DiFi-HRG tumor xenografts in nude mice to a greater extent than did treatment with either drug alone. Activation of HER2-HER3 signaling associated with the operation of a heregulin autocrine loop confers resistance to cetuximab, and patritumab is able to restore cetuximab sensitivity through inhibition of heregulin-induced HER3 activation.

Abstract 856: Combination of crizotinib and radiation in the treatment of ALK-positive and cetuximab-resistant lung cancer

Abstract Approximately 4% of patients with non-small cell lung carcinoma (NSCLC) carry the EML4-ALK chromosomal rearrangement. Crizotinib, a c-MET and ALK inhibitor, has shown significant therapeutic benefit as a single agent with FDA approval for the treatment of patients with ALK-positive NSCLC. Since radiation commonly plays a key role in lung cancer treatment, we sought to evaluate the potential of crizotinib as a radiation sensitizer and the role of ALK in cellular radiation sensitivity. We examined the effect of crizotinib in combination with radiation on ALK signaling, cell proliferation, cell cycle distribution and radiosensitivity in ALK-positive NSCLC cell lines H3122 and H2228 in vitro. We also examined the in-vivo effects of crizotinib in combination with radiation in H3122 and H2228 xenograft models. Crizotinib blocked phosphorylation of ALK and its downstream effectors and inhibited cell proliferation in a dose-dependent manner. The combination of crizotinib and radiation resulted in increased inhibition of cell growth. Although cells were sensitized to radiation by crizotinib, molecular knockdown of ALK by siRNA had only modest impact on cell radiosensitivity. Cell cycle analysis by flow cytometry showed that ALK-siRNA induced G1 arrest. Interestingly, low dose crizotinib induced G1 arrest, while doses above 2µM induced G2 arrest. Combination treatment induced arrest in G1 and G2 phases and decreased the cell population in S-phase. Moreover, in xenograft tumor models, we observed combined treatment to produce additive tumor growth inhibition over that observed with either drug or radiation alone. We further examined the capacity of crizotinib overcome cetuximab resistance in our established in vitro resistance models. Compared with parental cells, cetuximab-resistant cells showed cross-resistance to radiation. Crizotinib inhibited the growth of cetuximab-resistant cells and resensitized cells to radiation. Compared with single-agent treatment, the combination of crizotinib and erlotinib resulted in increased growth inhibition and further re-sensitization to radiation. These data suggest that crizotinib in combination with radiation can enhance the inhibitory effect on ALK-positive tumor cell growth in vitro and in-vivo and also suggest the potential value of using crizotinib in combination with other EGFR-targeting therapeutics. Ultimately however, the potential value of crizotinib as a radiation sensitizer requires systematic clinical investigation. Citation Format: Chunrong Li, Shyhmin Huang, Noah Walters, Eric A. Armstrong, Paul M. Harari. Combination of crizotinib and radiation in the treatment of ALK-positive and cetuximab-resistant lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 856. doi:10.1158/1538-7445.AM2014-856

Abstract 2474: Smad4 loss induces cetuximab resistance and increased metastatic potential in head and neck squamous cell carcinoma

Abstract Introduction: Cetuximab is a monoclonal antibody that increases survival in head and neck squamous cell carcinoma (HNSCC) patients when used in combination with radiation or chemotherapy. However, this EGFR targeted-agent only has modest activity when administered as a monotherapy and a majority of patients develop therapeutic resistance. Previous work has established the TGF-beta signaling pathway has a critical role in HNSCC development and progression. Modulation of this pathway, and more specifically loss of Smad4 expression, is a common means of developing drug resistance in many cancers. To determine the role of Smad4 loss in HNSCC development, metastasis, and cetuximab resistance, Smad4 was knocked down or reexpressed in HNSCC cell lines and evaluated in vitro and in vivo. Methods: Smad4 was knocked-down in SCC61 following stable introduction of Smad4-targeting shRNA. Using stable lentiviral transduction, Smad4 was reexpressed in FaDu, a HNSCC cell line which lacks de novo expression of Smad4. Following modulation, changes in cetuximab sensitivity were determined using Matrigel colony formation assays. The effects of Smad4 modulation on various signal transduction pathways were determined by Western blot analysis with total and phospho-specific antibodies. Changes in metastatic potential were evaluated using an orthotopic in vivo model. The subsequent development of lymph node metastases were monitored utilizing luciferin-based imaging. Key proteins associated with metastasis were also assayed using ELISA and protein antibody arrays. Results: Smad4 loss increased the growth of SCC61 and FaDu and Smad4 status correlated with cetuximab resistance in both cells. Western blot analysis demonstrated Smad4 loss activates JNK and MAPK signaling. Consequently, co-treatment of Smad4 knockdown cells with cetuximab and a JNK inhibitor reversed the previously observed therapeutic resistance. Smad4 knockdown also increased lymph node metastasis in the orthotopic in vivo model. Protein array analysis determined Smad4 knockdown increased the expression of amphiregulin, FGF2, and VEGF, pathways capable of promoting metastasis. Expression of these Smad4-sensitive targets was inhibited with the use of a MAPK inhibitor. Conclusion: We determined Smad4 loss enhances cetuximab resistance and in vivo metastasis development in HNSCC. Additionally, JNK and MAPK pathway activation play an important role in mediating this process. Our results suggest Smad4 downregulation plays a critical role for prognosis of HNSCC patients, and JNK and MAPK inhibition may contribute to improve therapeutic outcome of those patients. Citation Format: Hiroyuki Ozawa, Haixia Cheng, Elana J. Fertig, Jason D. Howard, Ana Markovic, Robert Hughes, Jimena Perez, Harry Quon, Christine H. Chung. Smad4 loss induces cetuximab resistance and increased metastatic potential in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2474. doi:10.1158/1538-7445.AM2014-2474

Abstract 4734: Activation of yap1 is significantly associated with poor prognosis and cetuximab resistance in colorectal cancer patients

Abstract Purpose: To investigate the clinical significance of activation of YAP1, a newly identified oncogene in colorectal cancer (CRC). Patients and Methods: A gene expression signature reflecting YAP1 activation was identified in CRC cells, and CRC patients were stratified into two groups according to this signature: activated YAP1 CRC (AYCC) or inactivated YAP1 CRC (IYCC). Stratified patients in six test cohorts were evaluated to determine the effect of the signature on CRC prognosis and response to treatment with cetuximab. Results: The activated YAP1 signature was associated with poor prognosis for CRC in four independent patient cohorts with stage I-III disease (total n = 1,028). In a multivariate analysis, the impact of the YAP1 signature on the disease-free survival rate was independent of other clinical variables [hazard ratio (HR), 1.63; 95% confidence interval (CI), 1.25-2.13; P &amp;lt; .001]. In patients with stage IV CRC, AYCC patients had a poorer disease control rate and progression-free survival duration after cetuximab-based monotherapy than did IYCC patients (HR, 1.82; 95% CI, 1.05-3.16; P = .03). In multivariate analysis, the effect of YAP1 activation on PFS was independent of KRAS mutation status and cetuximab-based therapy was effective only in IYCC patients without KRAS mutations. Conclusion: Activation of YAP1 is highly associated with poor prognosis for CRC and may be useful in identifying patients with CRC resistant to treatment with cetuximab. Citation Format: Keun-Wook Lee, Sung Sook Lee, Sang-Bae Kim, Yun-Yong Park, Bo Hwa Sohn, Hyun-Sung Lee, Ju-Seog Lee. Activation of yap1 is significantly associated with poor prognosis and cetuximab resistance in colorectal cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4734. doi:10.1158/1538-7445.AM2014-4734

Abstract 1844: Novel HER3 neutralizing antibody, patritumab abrogates cetuximab resistance mediated by a heregulin-autocrine loop in colorectal cancer

Abstract Purpose Acquired resistance to cetuximab is a major problem in the treatment of colorectal cancers. We previously showed that the HER3/HER4 ligand heregulin induced resistance to cetuximab by increasing the association between HER2 and HER3, and subsequent activation of downstream targets. We have now investigated strategies to overcome such resistance, using patritumab, a novel neutralizing HER3 antibody. Experimental design We established DiFi cells that stably express heregulin (DiFi-HRG cells) as a result of retroviral infection, and examined the antitumor effect of cetuximab, patritumab, and the combination of both in these cells in vitro and vivo. Results DiFi-HRG cells secreted large amount of heregulin and exhibited resistance to cetuximab. Cetuximab alone down-regulated ERK phosphorylation, whereas it had no effect on AKT phosphorylation, suggesting that continuous AKT activation underlain underlies resistance to cetuximab in these cells. In contrast, Patritumab in combination with cetuximab markedly reduced the level of phosphorylation of HER2 HER3 and AKT, as well as ERK, and induced apoptosis that was accompanied by upregulation of BIM and by downregulation of survivin in vitro. Furthermore, the combination therapy also markedly inhibited the growth of DiFi-HRG tumors in nude mice to a greater extent than did treatment with either drug alone. Conclusion Our data indicate that HER2/HER3 signaling activated by a heregulin-autocrine loop confers resistance to cetuximab, and that patritumab restored sensitivity to cetuximab by inhibition of HER3 phosphorylation that is stimulated by heregulin. Citation Format: Hisato Kawakami, Isamu Okamoto, Kimio Yonesaka, Kunio Okamoto, Kiyoko Kuwata, Yume Morita, Haruka Yamaguchi, Kazuto Nishio, Kazuhiko Nakagawa. Novel HER3 neutralizing antibody, patritumab abrogates cetuximab resistance mediated by a heregulin-autocrine loop in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1844. doi:10.1158/1538-7445.AM2014-1844

AXL mediates resistance to cetuximab therapy.

The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical outcome. In this study, we show that overexpression of the oncogenic receptor tyrosine kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where AXL was overexpressed, activated, and tightly associated with EGFR expression in cells resistant to cetuximab (Ctx(R) cells). Using RNAi methods and novel AXL-targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation, and MAPK signaling in Ctx(R) cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in Ctx(R) cells, creating a positive feedback loop that maintained EGFR activation by AXL. Cetuximab-sensitive parental cells were rendered resistant to cetuximab by stable overexpression of AXL or stimulation with EGFR ligands, the latter of which increased AXL activity and association with the EGFR. In tumor xenograft models, the development of resistance following prolonged treatment with cetuximab was associated with AXL hyperactivation and EGFR association. Furthermore, in an examination of patient-derived xenografts established from surgically resected HNSCCs, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL-targeting drugs to treat cetuximab-resistant cancers. Cancer Res; 74(18); 5152-64. ©2014 AACR.

EGFR-targeted TRAIL and a Smac mimetic synergize to overcome apoptosis resistance in KRAS mutant colorectal cancer cells.

TRAIL is a death receptor ligand that induces cell death preferentially in tumor cells. Recombinant soluble TRAIL, however, performs poorly as an anti-cancer therapeutic because oligomerization is required for potent biological activity. We previously generated a diabody format of tumor-targeted TRAIL termed DbαEGFR-scTRAIL, comprising single-stranded TRAIL molecules (scTRAIL) and the variable domains of a humanized variant of the EGFR blocking antibody Cetuximab. Here we define the bioactivity of DbαEGFR-scTRAIL with regard to both EGFR inhibition and TRAIL receptor activation in 3D cultures of Caco-2 colorectal cancer cells, which express wild-type K-Ras. Compared with conventional 2D cultures, Caco-2 cells displayed strongly enhanced sensitivity toward DbαEGFR-scTRAIL in these 3D cultures. We show that the antibody moiety of DbαEGFR-scTRAIL not only efficiently competed with ligand-induced EGFR function, but also determined the apoptotic response by specifically directing DbαEGFR-scTRAIL to EGFR-positive cells. To address how aberrantly activated K-Ras, which leads to Cetuximab resistance, affects DbαEGFR-scTRAIL sensitivity, we generated stable Caco-2tet cells inducibly expressing oncogenic K-RasG12V. In the presence of doxycycline, these cells showed increased resistance to DbαEGFR-scTRAIL, associated with the elevated expression of the anti-apoptotic proteins cIAP2, Bcl-xL and FlipS. Co-treatment of cells with the Smac mimetic SM83 restored the DbαEGFR-scTRAIL-induced apoptotic response. Importantly, this synergy between DbαEGFR-scTRAIL and SM83 also translated to 3D cultures of oncogenic K-Ras expressing HCT-116 and LoVo colorectal cancer cells. Our findings thus support the notion that DbαEGFR-scTRAIL therapy in combination with apoptosis-sensitizing agents may be promising for the treatment of EGFR-positive colorectal cancers, independently of their KRAS status.

Overcoming cetuximab resistance in HNSCC: The role of AURKB and DUSP proteins

Unraveling the underlying mechanisms of cetuximab resistance in head and neck squamous cell carcinoma (HNSCC) is of major importance as many tumors remain non-responsive or become resistant. Our microarray results suggest that “resistant” cells still exhibit RAS–MAPK pathway signaling contributing to drug resistance, as witnessed by low expression of DUSP5 and DUSP6, negative regulators of ERK1/2, and increased expression of AURKB, a key regulator of mitosis. Therefore, interrupting the RAS–MAPK pathway by an ERK1/2 inhibitor (apigenin) or an AURKB inhibitor (barasertib) might be a new strategy for overcoming cetuximab resistance in HNSCC.