Cetuximab-resistant Tumors Research Articles

Multivalent Rhamnose-Modified EGFR-Targeting Nanobody Gains Enhanced Innate Fc Effector Immunity and Overcomes Cetuximab Resistance via Recruitment of Endogenous Antibodies.

Cetuximab resistance is a significant challenge in cancer treatment, requiring the development of novel therapeutic strategies. In this study, a series of multivalent rhamnose (Rha)-modified nanobody conjugates are synthesized and their antitumor activities and their potential to overcome cetuximab resistance are investigated. Structure-activity relationship studies reveal that the multivalent conjugate D5, bearing sixteen Rha haptens, elicits the most potent innate fragment crystallizable (Fc) effector immunity in vitro and exhibits an excellent in vivo pharmacokinetics by recruiting endogenous antibodies. Notably, it is found that the optimal conjugate D5 represents a novel entity capable of reversing cetuximab-resistance induced by serine protease (PRSS). Moreover, in a xenograft mouse model, conjugate D5 exhibits significantly improved antitumor efficacy compared to unmodified nanobodies and cetuximab. The findings suggest that Rha-Nanobody (Nb) conjugates hold promise as a novel therapeutic strategy for the treatment of cetuximab-resistant tumors by enhancing the innate Fc effector immunity and enhancing the recruitment of endogenous antibodies to promote cancer cell clearance by innate immune cells.

Phosphoproteomics of patient-derived xenografts identifies targets and markers associated with sensitivity and resistance to EGFR blockade in colorectal cancer.

Epidermal growth factor receptor (EGFR) is a well-exploited therapeutic target in metastatic colorectal cancer (mCRC). Unfortunately, not all patients benefit from current EGFR inhibitors. Mass spectrometry-based proteomics and phosphoproteomics were performed on 30 genomically and pharmacologically characterized mCRC patient-derived xenografts (PDXs) to investigate the molecular basis of response to EGFR blockade and identify alternative drug targets to overcome resistance. Both the tyrosine and global phosphoproteome as well as the proteome harbored distinctive response signatures. We found that increased pathway activity related to mitogen-activated protein kinase (MAPK) inhibition and abundant tyrosine phosphorylation of cell junction proteins, such as CXADR and CLDN1/3, in sensitive tumors, whereas epithelial-mesenchymal transition and increased MAPK and AKT signaling were more prevalent in resistant tumors. Furthermore, the ranking of kinase activities in single samples confirmed the driver activity of ERBB2, EGFR, and MET in cetuximab-resistant tumors. This analysis also revealed high kinase activity of several members of the Src and ephrin kinase family in 2 CRC PDX models with genomically unexplained resistance. Inhibition of these hyperactive kinases, alone or in combination with cetuximab, resulted in growth inhibition of ex vivo PDX-derived organoids and in vivo PDXs. Together, these findings highlight the potential value of phosphoproteomics to improve our understanding of anti-EGFR treatment and response prediction in mCRC and bring to the forefront alternative drug targets in cetuximab-resistant tumors.

EGFR Alterations Influence the Cetuximab Treatment Response and c-MET Tyrosine-Kinase Inhibitor Sensitivity in Experimental Head and Neck Squamous Cell Carcinomas.

Background: Anti-EGFR antibody therapy is still one of the clinical choices in head and neck squamous cell carcinoma (HNSCC) patients, but the emergence of cetuximab resistance questioned its effectiveness and reduced its applicability. Although several possible reasons of resistance against the antibody treatment and alternative therapeutic proposals have been described (EGFR alterations, activation of other signaling pathways), there is no method to predict the effectiveness of anti-EGFR antibody treatments and to suggest novel therapeutics. Our study investigated the effect of EGFR R521K alteration on efficiency of cetuximab therapy of HNSCC cell lines and tried to find alternative therapeutic approaches against the resistant cells. Methods: After genetic characterization of HNSCC cells, we chose one wild type and one R521K+ cell line for in vitro proliferation and apoptosis tests, and in vivo animal models using different therapeutic agents. Results: Although the cetuximab treatment affected EGFR signalization in both cells, it did not alter in vitro cell proliferation or apoptosis. In vivo cetuximab therapy was also ineffective on R521K harboring tumor xenografts, while blocked the tumor growth of EGFR-wild type xenografts. Interestingly, the cetuximab-resistant R521K tumors were successfully treated with c-MET tyrosine kinase inhibitor SU11274. Conclusion: Our results suggest that HNSCC cell line expressing the R521K mutant form of EGFR does not respond well to cetuximab treatment in vitro or in vivo, but hopefully might be targeted by c-MET tyrosine kinase inhibitor treatment.

Combination of copanlisib with cetuximab improves tumor response in cetuximab-resistant patient-derived xenografts of head and neck cancer.

Despite recent advances, the treatment of head and neck squamous cell carcinoma (HNSCC) remains an area of high unmet medical need. HNSCC is frequently associated with either amplification or mutational changes in the PI3K pathway, making PI3K an attractive target particularly in cetuximab-resistant tumors. Here, we explored the antitumor activity of the selective, pan-class I PI3K inhibitor copanlisib with predominant activity towards PI3Kα and δ in monotherapy and in combination with cetuximab using a mouse clinical trial set-up with 33 patient-derived xenograft (PDX) models with known HPV and PI3K mutational status and available data on cetuximab sensitivity. Treatment with copanlisib alone resulted in moderate antitumor activity with 12/33 PDX models showing either tumor stabilization or regression. Combination treatment with copanlisib and cetuximab was superior to either of the monotherapies alone in the majority of the models (21/33), and the effect was particularly pronounced in cetuximab-resistant tumors (14/16). While no correlation was observed between PI3K mutation status and response to either cetuximab or copanlisib, increased PI3K signaling activity evaluated through gene expression profiling showed a positive correlation with response to copanlisib. Together, these data support further investigation of PI3K inhibition in HNSCC and suggests gene expression patterns associated with PI3K signaling as a potential biomarker for predicting treatment responses.

Abstract 4834: Effective treatment of squamous cell carcinoma of the head and neck (HNSCC) using a combined regimen of tipifarnib and cetuximab

Abstract Background: H-RAS driven carcinogenesis has been reported in many types of cancer including squamous cell carcinoma of the head and neck (HNSCC). The mutation rate of H-RAS in HNSCC is 4 - 8%. Tipifarnib is a potent and highly selective inhibitor of farnesyltransferase (FT). It is known that H-RAS, but not K-RAS and N-RAS, is delocalized into cytoplasm and inactivated by farnesyltransferase inhibitors (FTI), such as tipifarnib. Tipifarnib has demonstrated proof of concept activity in H-RAS mutant HNSCC in an ongoing clinical trial (NCT02383927). In this study, we examined H-RAS associated signaling pathway in HNSCC cell lines and PDX models to see if inhibition of wild-type HRAS signaling could re-sensitize cetuximab resistant cells and tumors to inhibition by cetuximab. UMSCC47 and SCC1-C are both H-RAS wild-type HNSCC cell lines, and SCC1-C is resistant to EGFR targeted therapy. Methods: In vitro cell growth with and without tipifarnib and/or cetuximab treatments were examined by Sulforhodamine B (SRB) and colony formation assay. Patient derived xenograft (PDX) models were then used to study the efficacy of the tipifarnib treatment as a single agent or in combination with cetuximab. Western blot analyses were performed to verify the effect of these treatments on EGFR/ERK/AKT signaling pathways. Results: Our results showed that tipifarnib could inhibit HNSCC growth, both in vitro and in the PDX model in a dose-dependent manner (control vs the low dose of tipifarnib, p = 0.02; control vs the high dose of tipifarnib, p = 0.001). However, the combined treatment with cetuximab significantly enhanced tipifarnib efficacy in the H-RAS wild type setting, particularly in the PDX model (control vs tipifarnib, p = 0.123; control vs cetuximab, p = 0.022; control vs the combination, p = 0.010). Western blot analyses showed that neither ERK nor AKT phosphorylation was effectively reduced after a prolonged treatment (48 hours) with tipifarnib alone. However, the addition of cetuximab reduced pERK. Conclusions: Our study demonstrates that cetuximab enhances the anti-tumor effect of tipifarnib in the H-RAS wild type setting through ERK inhibition. This study supports the rationale for combining tipifarnib with EGFR inhibitors as a possible effective therapeutic approach in HNSCC. Citation Format: Lihua Shu, Dongsheng Wang, Sreenivas Nannapaneni, Dong M. Shin, Nabil F. Saba, Georgia Z. Chen. Effective treatment of squamous cell carcinoma of the head and neck (HNSCC) using a combined regimen of tipifarnib and cetuximab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4834.

Preclinical efficacy of copanlisib in cetuximab sensitive and resistant tumors of HNSCC.

6031 Background: Copanlisib is a highly selective, pan-class I PI3K inhibitor with preferential activity against the p110α and p110δ isoforms that lead to downregulation of PI3K signaling. Copanlisib has been approved for the treatment of follicular lymphoma in the US. Here, we explored the anti-tumor activity of copanlisib in head and neck squamous cell carcinoma (HNSCC), where Pi3K signaling has been defined as alternate signaling in cetuximab resistant tumors. Further, TCGA data show up to 56% of HNSCC display either amplification or mutational changes in the Pi3K pathway making Pi3K an attractive target. Methods: Using a mouse-clinical trial set-up we profiled 20 patient derived HNSCC xenograft models for their sensitivity to cetuximab or copanlisib as single agent as well as in combination. Models were selected from our HNSCC PDX platform based on Pi3K mutational status, with 6 models harboring hot spot mutations, HPV positivity (n=3) and/or cetuximab resistance based on previous drug screenings (n=12). Treatment response was defined as tumor regression, stabilization or progression expressed as relative tumor volume (RTV) after 3 weeks of treatment: RTV<0,7 responder, RTV>1,2 progressor. Results: Copanlisib single agent treatment resulted in moderate activity with 5 responders (25%). In cetuximab resistant tumors (n=12) combined treatment led to an improved tumor response in 75% (n=9) whereas 41% (n=5) resulted in tumor control. Pi3KCA mutation was not predictive for treatment response to either cetuximab or copanlisib. No PTEN mutation was detected in the selected cohort. Increased Pi3K signaling activity evaluated through gene expression profiling and computed with GSEA pathway analyses was positively correlated with response. Conclusions: The anti-tumor responses observed in monotherapy or in combination treatment support further investigation for the potential of PI3K inhibition in HNSCC with high expression of Pi3K pathway signature as a potential predictive biomarker.

Therapeutic efficacy of SYM004, a mixture of two anti-EGFR antibodies in human colorectal cancer with acquired resistance to cetuximab and MET activation.

PurposeCetuximab and panitumumab have an effective therapeutic response in a subset of RAS Wild-Type (WT) metastatic colorectal cancers (mCRCs). Despite molecular-driven selection, all patients do not respond to epidermal growth factor receptor (EGFR) inhibitors and the onset of secondary resistance limits their clinical benefit.Experimental DesignWe tested, in vitro and in vivo, the effect of SYM004, a 1:1 mixture of two recombinant human-mouse chimeric monoclonal antibodies (mAbs) directed against non-overlapping epitopes of the EGFR, on CRC models with acquired resistance to cetuximab.ResultsSYM004 showed a potent growth inhibitory effect in CRC cell lines with acquired resistance to cetuximab and MET activation. SYM004 treatment determined a significant induction of apoptosis and a strong inhibition of MET, AKT and MAPK phosphorilation in these resistant models. The data may further suggest SYM004 -driven induced internalization and degradation of the antibody-receptor complex, which prevents cross-interaction between EGFR and MET even in the presence of TGFα. Moreover, in vivo xenograft studies demonstrated that SYM004 has stronger antitumor activity than cetuximab in CRC models. Importantly, in the current work we observed a response to therapy in all cetuximab resistant tumors mice treated with SYM004. More importantly, four out of seven mice continue to respond to SYM004 after 30 weeks of treatment underling the prolonged effect of the drug.ConclusionThese results suggest that the treatment with SYM004 could be a strategy to overcome acquired resistance to first generation of anti-EGFR therapies in mCRC as a result of MET activation.

Rationally co-targeting divergent pathways in KRAS wild-type colorectal cancers by CANscript technology reveals tumor dependence on Notch and Erbb2

KRAS mutation status can distinguish between metastatic colorectal carcinoma (mCRC) patients who may benefit from therapies that target the epidermal growth factor receptor (EGFR), such as cetuximab. However, patients whose tumors harbor mutant KRAS (codons 12/13, 61 and 146) are often excluded from EGFR-targeted regimens, while other patients with wild type KRAS will sometimes respond favorably to these same drugs. These conflicting observations suggest that a more robust approach to individualize therapy may enable greater frequency of positive clinical outcome for mCRC patients. Here, we utilized alive tumor tissues in ex-vivo platform termed CANscript, which preserves the native tumor heterogeneity, in order to interrogate the antitumor effects of EGFR-targeted drugs in mCRC (n = 40). We demonstrated that, irrespective of KRAS status, cetuximab did not induce an antitumor response in a majority of patient tumors. In the subset of non-responsive tumors, data showed that expression levels of EGFR ligands contributed to a mechanism of resistance. Transcriptomic and phosphoproteomic profiling revealed deregulation of multiple pathways, significantly the Notch and Erbb2. Targeting these nodes concurrently resulted in antitumor efficacy in a majority of cetuximab-resistant tumors. These findings highlight the importance of integrating molecular profile and functional testing tools for optimization of alternate strategies in resistant population.

Afatinib plus Cetuximab Delays Resistance Compared to Single-Agent Erlotinib or Afatinib in Mouse Models of TKI-Naïve EGFR L858R-Induced Lung Adenocarcinoma.

The EGFR tyrosine kinase inhibitors (TKIs), erlotinib and afatinib, have transformed the treatment of advanced EGFR-mutant lung adenocarcinoma. However, almost all patients who respond develop acquired resistance on average approximately 1 year after starting therapy. Resistance is commonly due to a secondary mutation in EGFR (EGFR(T790M)). We previously found that the combination of the EGFR TKI afatinib and the EGFR antibody cetuximab could overcome EGFR(T790M)-mediated resistance in preclinical models. This combination has shown a 29% response rate in a clinical trial in patients with acquired resistance to first-generation TKIs. An outstanding question is whether this regimen is beneficial when used as first-line therapy. Using mouse models of EGFR-mutant lung cancer, we tested whether the combination of afatinib plus cetuximab delivered upfront to mice with TKI-naïve EGFR(L858R)-induced lung adenocarcinomas delayed tumor relapse and drug-resistance compared with single-agent TKIs. Afatinib plus cetuximab markedly delayed the time to relapse and incidence of drug-resistant tumors, which occurred in only 63.6% of the mice, in contrast to erlotinib or afatinib treatment where 100% of mice developed resistance. Mechanisms of tumor escape observed in afatinib plus cetuximab resistant tumors include the EGFR(T790M) mutation and Kras mutations. Experiments in cell lines and xenografts confirmed that the afatinib plus cetuximab combination does not suppress the emergence of EGFR(T790M). These results highlight the potential of afatinib plus cetuximab as an effective treatment strategy for patients with TKI-naïve EGFR-mutant lung cancer and indicate that clinical trial development in this area is warranted.

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.

Safety, pharmacology, and preliminary clinical activity of MM-151: An oligocolnal anti-EGFR theraputic in patients with cetuximab-resistant CRC and other refractory solid tumors.

647 Background: MM-151 is an oligoclonal anti-EGFR antibody combination designed to provide enhanced targeting of the EGFR network. This work describes the first in human experience of MM-151 alone and in combination with irinotecan. Methods: A standard 3+3 design was used to assess safety and tolerability of MM-151 monotherapy (QW, Q2W, Q3W) and MM-151 (QW) in combination with irinotecan 180mg/m2 (Q2W). A monotherapy expansion cohort has begun in cetuximab-resistant CRC. Results: Results presented are based on preliminary data as of September 2, 2014, for MM-151 monotherapy (n=65; QW/Q2W) and MM-151 in combination with irinotecan (n=10). The most common tumor types were CRC (29 [39%]), NSCLC (9 [12%]), HNSCC (8 [11%]). The monotherapy RP2D has been defined as 10.5 mg/kg QW and dosing continues at 18 mg/kg Q2W. Most adverse events were CTCAE grades 1 and 2. The most common AEs were toxicities related to the EGFR-pathway, including rash (70%), hypomagnesemia (24%), fatigue, dry skin, and diarrhea (21%). IRR was initially observed at high frequencies; however, standardized pre-medication and a modified infusion schedule significantly decreased this frequency. At doses >9 mg/kg QW, trough total antibody levels at steady state were in the expected therapeutic range. Of the CRC patients enrolled, 3 experienced Partial Responses (PRs) per RECIST criteria and a total of 8 (31%) pts had SD for >4 months. Initial biomarker data suggests particular clinical activity in triple-wildtype, EGFR-ligand positive CRC patients. Combination therapy with irinotecan continues with MM-151 at 9 mg/kg QW and 3 PRs have been observed (CRC, HNSCC, melanoma). Initial safety data is consistent with known toxicities of MM-151 and irinotecan. Preliminary results from the monotherapy expansion cohort (10.5 mg/kg QW) in CTX-refractory CRC will also be presented. Conclusions: These results demonstrate that MM-151 monotherapy has an acceptable tolerability profile and objective clinical activity alone and in combination with irinotecan. Results also support further clinical evaluation in triple-wildtype, EGFR-ligand positive CRC and other EGFR dependent cancers. Clinical trial information: NCT01520389.

Enhancement of tumor uptake and therapeutic efficacy of EGFR-targeted antibody cetuximab and antibody-drug conjugates by cholesterol sequestration.

Cetuximab, a monoclonal antibody (mAb) targeting the epidermal growth factor receptor (EGFR), has been intensively investigated as a promising cancer treatment strategy. The specific mechanism of cetuximab endocytosis and its influence on cetuximab uptake, biodistribution and efficacy still remain elusive. Recently, statins have been reported to synergize with EGFR-targeting agents. Our prior work established that nystatin, a cholesterol-sequestering antifungal drug, facilitates endocytosis via the clathrin-dependent pathway. This study aimed to investigate whether nystatin regulates the uptake and efficacy of cetuximab and cetuximab-based antibody-drug conjugates (cetuximab-ADCs). In vitro and in vivo efficacies of nystatin on the uptake and activity of cetuximab/cetuximab-ADCs were studied in multiple human carcinoma cell lines and xenograft models, respectively. We identified that cholesterol sequestration by nystatin enhanced cetuximab internalization in EGFR-positive carcinoma cells by regulating EGFR trafficking/turnover and facilitating a switch from lipid rafts to clathrin-mediated endocytosis. Combination treatment with cetuximab and nystatin selectively increased cetuximab uptake by tumor tissues, translating into potentiated antitumor efficacy of cetuximab in vivo (A431 and A549 tumors). Nystatin-enhanced internalization of cetuximab further improved the uptake and potency of cetuximab-doxorubicin and cetuximab-methotrexate conjugates in EGFR-positive cetuximab-resistant tumors. Combination therapy with nystatin plus either cetuximab or cetuximab-ADC further prolonged animal survival and significantly suppressed tumor growth, as compared with single-agent cetuximab or cetuximab-ADC. In summary, our results identify a novel mechanism whereby cholesterol sequestration enhances the uptake of EGFR-targeting mAb and ADCs, therefore providing preclinical proof-of-concept that combination with nystatin can potentiate the delivery and efficacy of these EGFR-targeted agents.

Abstract C90: Dependence of afatinib and cetuximab resistant lung adenocarcinomas on mTOR signaling.

Abstract Lung adenocarcinomas harboring mutations in the tyrosine kinase domain of the EGFR are sensitive to tyrosine kinase inhibitors (TKIs) erlotinib or gefitinib. Despite a ∼70% response rate to these agents, patients almost inevitably develop resistance on average within a year of starting drug treatment. In 50% of cases, acquired resistance to EGFR TKIs is due to the emergence of a secondary mutation in the tyrosine kinase domain of the receptor (the T790M mutation). Additional less common mechanisms of resistance include activation of bypass signaling pathways via amplification of other receptor tyrosine kinases (RTKs) like MET and HER2 or mutations in genes encoding downstream signaling components such as PIK3CA or BRAF. In rare instances, tumors biopsied at progression show phenotypic transformations such as epithelial-to-mesenchymal transition (EMT) or neuroendocrine differentiation. We previously generated transgenic mice that develop EGFRL858R+T790M-induced lung adenocarcinomas and showed that T790M-mediated resistance could be overcome using a combination of a second generation TKI BIBW-2992 (afatinib) with the anti-EGFR antibody, cetuximab. This preclinical study prompted a Phase IB/II clinical trial testing this drug combination in patients with progressive disease after erlotinib or gefitinib treatment that is showing a promising 30% response rate. Unfortunately patients eventually develop disease progression on this drug combination and the mechanisms of resistance to afatinib+cetuximab are currently unknown. Here, we used an intermittent dosing strategy, previously used to generate erlotinib-resistant tumors in mice, to generate afatinib+cetuximab resistant tumors in xenograft and transgenic mouse models of EGFR mutant lung cancer. In these models, afatinib+cetuximab resistant tumors lacked detectable mutations in the EGFR transgene, the ERBB2 kinase domain or KRAS. Molecular analysis of resistant tumors revealed high levels of activation of the mTOR signaling pathway. Consistent with these findings, two patients with afatinib+cetuximab-resistant tumors exhibited alterations in genes (TSC1 and NF2) that activate the mTOR pathway. In cell culture and in mouse models, such resistance can be overcome by addition of the mTOR pathway inhibitor, rapamycin. These studies are the first to demonstrate mechanisms of acquired resistance to dual inhibition of EGFR in EGFR mutant lung cancer and provide new insight into the biology of this subset of lung cancers with immediate therapeutic implications for patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C90. Citation Format: Valentina Pirazzoli, Caroline Nebhan, Xiaoling Song, Zenta Walter, Guoping Cai, Anna Wurtz, Zhongming Zhao, Elisa Elisa de Stanchina, Leora Horn, David Carbone, Philip J. Stevens, Vincent Miller, Scott Gettinger, William Pao, Katerina Politi. Dependence of afatinib and cetuximab resistant lung adenocarcinomas on mTOR signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C90.

Abstract 933: Modeling acquired resistance to EGFR-directed therapies in mouse models of lung cancer.

Abstract Seventy percent of patients with Epidermal Growth Factor Receptor (EGFR) mutant lung cancer respond to treatment with the tyrosine kinase inhibitors (TKIs) erlotinib or gefitinib. Despite this high response rate, patients almost inevitably develop resistance to these drugs on average within a year of starting drug treatment. Acquired resistance to EGFR TKIs is most commonly due to the emergence of a secondary mutation (T790M) in EGFR (in 50% of cases), amplification of the genes encoding the ERBB2 and MET receptor tyrosine kinases in 12 and 5% of cases, respectively, and phenotypic transformation of the adenocarcinomas to small cell lung cancer (5% of cases). Previously, in an effort to develop strategies to overcome T790M-mediated resistance, we generated tetracycline-inducible transgenic mice that express the EGFRL858R+T790M in the lung epithelium. Upon administration of doxycycline these mice develop lung adenocarcinomas that are resistant to TKIs. However, the combination of the irreversible TKI afatinib and the EGFR antibody cetuximab showed dramatic responses in these transgenic mice. These preclinical studies led to a clinical trial of these agents, which has showing a promising 30% response rate in patients with EGFR mutant tumors resistant to TKIs. However, tumors also acquire resistance to this drug combination and the mechanisms of resistance to afatinib+cetuximab are currently unknown. We set out to identify these mechanisms using xenograft and transgenic mouse models of EGFR mutant lung cancer. Transgenic mice with EGFRL858R+T790M-induced tumors were treated with afatinib+cetuximab using an intermittent dosing strategy that we had previously used to generate erlotinib-resistant tumors in mice with EGFRL858R and EGFRDEL-induced tumors. 75% of mice develop afatinib+cetuximab resistant tumors after three month-long rounds of treatment. The same treatment strategy applied to xenografts harboring subcutaneous tumors induced by EGFRDEL+T790M gave rise to resistant tumors in 20% of cases. Analysis of the afatinib+cetuximab resistant tumors performed to date has not revealed additional mutations in the EGFR transgene, the ERBB2 kinase domain or KRAS. Additional sequencing studies and examination of signaling pathway alterations in the resistant tumors are ongoing. Uncovering mechanisms of resistance to this drug combination will allow the development of strategies to treat tumors that acquire resistance to EGFR-directed therapies. Citation Format: Valentina Pirazzoli, Elisa de Stanchina, Jungfeng Xia, Zhongming Zhao, William Pao, Katerina A. Politi. Modeling acquired resistance to EGFR-directed therapies in mouse models of lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 933. doi:10.1158/1538-7445.AM2013-933

Abstract 5646: Overcoming acquired resistance to cetuximab by dual targeting of HER family members using antibody based therapy.

Abstract The EGFR is recognized as a key mediator of proliferation and progression in many human tumors and strategies to inhibit EGFR signaling have emerged as highly promising cancer therapy approaches. However, many tumors do not show response to EGFR inhibition and some of the responders eventually manifest resistance to treatment. In an effort to examine mechanisms of acquired resistance to anti-EGFR monoclonal antibody we developed a series of cetuximab-resistant clones using the NSCLC line NCI-H226. During efforts to elucidate molecular pathways that are involved in acquired resistance to cetuximab we found that 1) EGFR ubiquitination and degradation is altered in cetuximab-resistant cells resulting in increased steady-state expression of the EGFR and 2) enhanced signaling from the EGFR to HER3 and the PI(3)K/Akt pathway indicating that activation of HER3 may lead to escape from cetuximab therapy. To investigate the role of HER3 in acquired resistance to cetuximab we used both RNAi and targeted approaches using the anit-HER3 antibody U3-1287/AMG888. siRNA directed against HER3 indicated that resistant cells had a strong dependency on HER3. Further, targeting HER3 using U3-1287/AMG888 showed a dose-dependent anti-proliferative response in resistant cells. In studies using siRNA against the EGFR and HER3 resulted in robust inhibition of cell proliferation than did silencing of either receptor alone in resistant cells. Comparable effects on cell proliferation were observed after combinatorial treatment of cells by the EGFR antibody cetuximab and the HER3 antibody U3-1287/AMG888. In addition, combined EGFR and HER3 inhibition by either siRNAs or antibodies decreased the phosphorylation of downstream signaling pathways AKT and MAPK. Annexin-V binding assays showed a significant increase in apoptosis in U3-1287/AMG888 plus cetuximab treated cells as compared to either agent alone. To determine if U3-1287/AMG888 could have therapeutic benefit as a combination treatment with cetuximab in vivo, mice harboring NCI-H226 xenografts will be treated with cetuximab for approximately 70-100 days until cetuximab-resistant tumors develop. At this time mice with resistant tumors will be divided into two groups that will be treated with both cetuximab and U3-1287/AMG888 or cetuximab alone. The work presented herein suggests that cells with acquired resistance to cetuximab maintain their dependence on EGFR and HER3 signaling via the AKT and MAPK pathways. Citation Format: Mari Iida, Toni M. Brand, Megan M. Starr, Neha Luthar, Matthew J. Wleklinski, Deric L. Wheeler. Overcoming acquired resistance to cetuximab by dual targeting of HER family members using antibody based therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5646. doi:10.1158/1538-7445.AM2013-5646

Dual Kinase Inhibition of EGFR and HER2 Overcomes Resistance to Cetuximab in a Novel In Vivo Model of Acquired Cetuximab Resistance

Acquired resistance to cetuximab, a chimeric epidermal growth factor receptor (EGFR)-targeting monoclonal antibody, is a widespread problem in the treatment of solid tumors. The paucity of preclinical models has limited investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. The purpose of this study was to generate cetuximab-resistant tumors in vivo to characterize mechanisms of acquired resistance. We generated cetuximab-resistant clones from a cetuximab-sensitive bladder cancer cell line in vivo by exposing cetuximab-sensitive xenografts to increasing concentrations of cetuximab, followed by validation of the resistant phenotype in vivo and in vitro using invasion assays. A candidate-based approach was used to examine the role of HER2 on mediating cetuximab resistance both in vitro and in vivo. We generated a novel model of cetuximab resistance, and, for the first time in the context of EGFR-inhibitor resistance, we identified increased phosphorylation of a C-terminal fragment of HER2 (611-CTF) in cetuximab-resistant cells. Afatinib (BIBW-2992), an irreversible kinase inhibitor targeting EGFR and HER2, successfully inhibited growth of the cetuximab-resistant cells in vitro. When afatinib was combined with cetuximab in vivo, we observed an additive growth inhibitory effect in cetuximab-resistant xenografts. These data suggest that the use of dual EGFR-HER2 kinase inhibitors can enhance responses to cetuximab, perhaps in part due to downregulation of 611-CTF. This study conducted in a novel in vivo model provides a mechanistic rationale for ongoing phase I clinical trials using this combination treatment modality.

Erlotinib is a viable treatment for tumors with acquired resistance to cetuximab

The epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase (RTK) and is recognized as a key mediator of tumorigenesis in many human tumors. Currently there are five EGFR inhibitors used in oncology, two monoclonal antibodies (panitumumab, and cetuximab) and three tyrosine kinase inhibitors (erlotinib, gefitinib, and lapatinib). Both strategies of EGFR inhibition have demonstrated clinical successes, however many tumors remain non-responsive or acquire resistance during therapy. To explore potential molecular mechanisms of acquired resistance to cetuximab we previously established a series of cetuximab-resistant clones by chronically exposing the NCI-H226 NSCLC cell line to escalating doses of cetuximab. Cetuximab-resistant clones exhibited a dramatic increase in steady-state expression of EGFR, HER2, and HER3 receptors as well as increased signaling through the MAPK and AKT pathways. RNAi studies demonstrated dependence of cetuximab-resistant clones on the EGFR signaling network. These findings prompted investigation on whether or not cells with acquired resistance to cetuximab would be sensitive to the EGFR targeted TKI erlotinib. In vitro, erlotinib was able to decrease signaling through the EGFR axis, decrease cellular proliferation, and induce apoptosis. To determine if erlotinib could have therapeutic benefit in vivo, we established cetuximab-resistant NCI-H226 mouse xenografts, and subsequently treated them with erlotinib. Mice harboring cetuximab-resistant tumors treated with erlotinib exhibited either a tumor regression or growth delay as compared to vehicle controls. Analysis of the erlotinib treated tumors demonstrated a decrease in cell proliferation and increase rates of apoptosis. The work presented herein suggests that 1) cells with acquired resistance to cetuximab maintain their dependence on EGFR and 2) tumors developing resistance to cetuximab can benefit from subsequent treatment with erlotinib, providing rationale for its use in the setting of cetuximab resistance.

Abstract 726: Erlotinib overcomes acquired resistance to cetuximab

Abstract The epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase (RTK) and is recognized as a key mediator of proliferation and progression in many human epithelial tumors. Following more than 20 years of preclinical development, five EGFR inhibitors, two monoclonal antibodies and three small molecule tyrosine kinase inhibitors (TKIs), have gained FDA approval in oncology (panitumumab, cetuximab, erlotinib, gefitinib and lapatinib). Both strategies of EGFR inhibition have demonstrated clinical successes. However, many tumors do not show response to EGFR inhibition and some of the responders eventually manifest resistance to the treatment. The underlying mechanisms of intrinsic and acquired resistance to EGFR inhibitors remain an intense area of scientific and clinical investigation. To explore potential molecular mechanisms of acquired resistance to cetuximab we established a series of cetuximab-resistant clones by chronically exposing the NCI-H226 NSCLC cell line to cetuximab. Cetuximab-resistant clones exhibited a dramatic increase in the steady-state expression of EGFR, due to alterations in trafficking and endocytosis as compared to parental controls. This increase in steady-state expression of the EGFR led to constitutive activation of HER2 and HER3 and downstream signals mediated by MAPK and AKT. Cells with acquired resistance to cetuximab remained dependent on EGFR signaling as shown by RNAi studies. This finding prompted investigation on whether or not cells with acquired resistance to cetuximab would be sensitive to erlotinib, an EGFR directed TKI. In vitro, erlotinib was able to decrease AKT and MAPK activation and subsequent cell proliferation measured by BrdU incorporation. Additionally, erlotinib treatment induced apoptosis in cetuximab resistant cells as measured by Annexin V analysis. To determine if erlotinib could have therapeutic benefit in vivo, we treated cetuximab sensitive parental lines, H226, with cetuximab in mouse xenograft studies. Mice harboring tumors were treated for approximately 70-150 days before resistant tumors manifested. At this time a series of mice with resistant tumors were treated with erlotinib for 15-30 days. Mice treated with erlotinib exhibited either a regression or tumor growth delay as compared to controls maintained on cetuximab. Analysis of the erlotinib treated tumors demonstrated a decrease in cell proliferation as measured by Ki67 and higher rates of apoptosis, as measured by TUNEL assays when compared to tumors maintained on cetuximab treatment. The work presented herein suggest that 1) cells with acquired resistance to cetuximab maintain their dependence on EGFR signaling via the MAPK and AKT pathways and 2) tumors developing resistance to cetuximab can be treated with erlotinib. This work may provide data strengthening the rationale for the use of erlotinib to treat patients with cetuximab resistant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 726. doi:10.1158/1538-7445.AM2011-726

Nuclear EGFR contributes to acquired resistance to cetuximab

Epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase involved in the etiology of several human cancers. Cetuximab is an EGFR-blocking antibody that has been approved for the treatment of patients with head and neck squamous cell carcinoma and metastatic colorectal cancer. Previous reports have shown that EGFR translocation to the nucleus is associated with cell proliferation. Here we investigated mechanisms of acquired resistance to cetuximab using a model derived from the non-small cell lung cancer line H226. We demonstrated that cetuximab-resistant cells overexpress HER family ligands including epidermal growth factor (EGF), amphiregulin, heparin-binding EGF and beta-cellulin. Overexpression of these ligands is associated with the nuclear translocation of the EGFR and this process was mediated by the Src family kinases (SFK). Treatment of cetuximab-resistant cells with the SFK inhibitor, dasatinib, resulted in loss of nuclear EGFR, increased membrane expression of the EGFR and resensitization to cetuximab. In addition, expression of a nuclear localization sequence-tagged EGFR in cetuximab-sensitive cells increased resistance to cetuximab both in vitro and in mouse xenografts. Collectively, these data suggest that nuclear expression of EGFR may be an important molecular determinant of resistance to cetuximab therapy and provides a rationale for investigating nuclear EGFR as a biomarker for cetuximab response. Further, these data suggest a rationale for the design of clinical trials that examine the value of treating patients with cetuximab-resistant tumors with inhibitors of SFKs in combination with cetuximab.

TLR9 agonist acts by different mechanisms synergizing with bevacizumab in sensitive and cetuximab-resistant colon cancer xenografts

Synthetic agonists of Toll-like receptor 9 (TLR9), a class of agents that induce specific immune response, exhibit antitumor activity and are currently being investigated in cancer patients. Intriguingly, their mechanisms of action on tumor growth and angiogenesis are still incompletely understood. We recently discovered that a synthetic agonist of TLR9, immune modulatory oligonucleotide (IMO), acts by impairing epidermal growth factor receptor (EGFR) signaling and potently synergizes with anti-EGFR antibody cetuximab in GEO human colon cancer xenografts, whereas it is ineffective in VEGF-overexpressing cetuximab-resistant GEO cetuximab-resistant (GEO-CR) tumors. VEGF is activated by EGFR, and its overexpression causes resistance to EGFR inhibitors. Therefore, we used IMO and the anti-VEGF antibody bevacizumab as tools to study IMO's role on EGFR and angiogenesis and to explore its therapeutic potential in GEO, LS174T, and GEO-CR cancer xenografts. We found that IMO enhances the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of cetuximab, that bevacizumab has no ADCC, and IMO is unable to enhance it. Nevertheless, the IMO-plus-bevacizumab combination synergistically inhibits the growth of GEO and LS174T as well as of GEO-CR tumors, preceded by inhibition of signaling protein expression, microvessel formation, and human, but not murine, VEGF secretion. Moreover, IMO inhibited the growth, adhesion, migration, and capillary formation of VEGF-stimulated endothelial cells. The antitumor activity was irrespective of the TLR9 expression on tumor cells. These studies demonstrate that synthetic agonists of TLR9 interfere with growth and angiogenesis also by EGFR- and ADCC-independent mechanisms affecting endothelial cell functions and provide a strong rationale to combine IMO with bevacizumab and EGFR inhibitory drugs in colon cancer patients.