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Cetuximab, the FDA-approved anti-EGFR antibody for head and neck squamous cell carcinoma (HNSCC), has displayed limited efficacy due to the emergence of intrinsic and acquired resistance. We and others have demonstrated that cetuximab resistance in HNSCC is driven by alternative receptor tyrosine kinases (RTK), including HER3, MET, and AXL. In an effort to overcome cetuximab resistance and circumvent toxicities associated with the administration of multiple RTK inhibitors, we sought to identify a common molecular target that regulates expression of multiple RTK. Bromodomain-containing protein-4 (BRD4) has been shown to regulate the transcription of various RTK in the context of resistance to PI3K and HER2 inhibition in breast cancer models. We hypothesized that, in HNSCC, targeting BRD4 could overcome cetuximab resistance by depleting alternative RTK expression. We generated independent models of cetuximab resistance in HNSCC cell lines and interrogated their RTK and BRD4 expression profiles. Cetuximab-resistant clones displayed increased expression and activation of several RTK, such as MET and AXL, as well as an increased percentage of BRD4-expressing cells. Both genetic and pharmacologic inhibition of BRD4 abrogated cell viability in models of acquired and intrinsic cetuximab resistance and was associated with a robust decrease in alternative RTK expression by cetuximab. Combined treatment with cetuximab and bromodomain inhibitor JQ1 significantly delayed acquired resistance and RTK upregulation in patient-derived xenograft models of HNSCC. These findings indicate that the combination of cetuximab and bromodomain inhibition may be a promising therapeutic strategy for patients with HNSCC. Significance: Inhibition of bromodomain protein BRD4 represents a potential therapeutic strategy to circumvent the toxicities and financial burden of targeting the multiple receptor tyrosine kinases that drive cetuximab resistance in HNSCC and NSCLC. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4331/F1.large.jpg. Cancer Res; 78(15); 4331–43. ©2018 AACR.

Cetuximab, an antibody against the EGFR, has shown efficacy in treating head and neck squamous cell carcinoma (HNSCC), metastatic colorectal cancer, and non–small cell lung cancer (NSCLC). Despite the clinical success of cetuximab, many patients do not respond to cetuximab. Furthermore, virtually all patients who do initially respond become refractory, highlighting both intrinsic and acquired resistance to cetuximab as significant clinical problems. To understand mechanistically how cancerous cells acquire resistance, we previously developed models of acquired resistance using the H226 NSCLC and UM-SCC1 HNSCC cell lines. Cetuximab-resistant clones showed a robust upregulation and dependency on the HER family receptors EGFR, HER2, and HER3. Here, we examined pan-HER, a mixture of six antibodies targeting these receptors on cetuximab-resistant clones. In cells exhibiting acquired or intrinsic resistance to cetuximab, pan-HER treatment decreased all three receptors' protein levels and downstream activation of AKT and MAPK. This correlated with decreased cell proliferation in cetuximab-resistant clones. To determine whether pan-HER had a therapeutic benefit in vivo, we established de novo cetuximab-resistant mouse xenografts and treated resistant tumors with pan-HER. This regimen resulted in a superior growth delay of cetuximab-resistant xenografts compared with mice continued on cetuximab. Furthermore, intrinsically cetuximab-resistant HNSCC patient-derived xenograft tumors treated with pan-HER exhibited significant growth delay compared with vehicle/cetuximab controls. These results suggest that targeting multiple HER family receptors simultaneously with pan-HER is a promising treatment strategy for tumors displaying intrinsic or acquired resistance to cetuximab. Mol Cancer Ther; 15(9); 2175–86. ©2016 AACR.

The epidermal growth factor receptor (EGFR) is a therapeutic target in patients with various cancers. Unfortunately, resistance to EGFR-targeted therapeutics is common. Previous studies identified two mechanisms of resistance to the EGFR monoclonal antibody cetuximab. Nuclear translocation of EGFR bypasses the inhibitory effects of cetuximab, and the receptor tyrosine kinase AXL mediates cetuximab resistance by maintaining EGFR activation and downstream signaling. Thus, we hypothesized that AXL mediated the nuclear translocation of EGFR in the setting of cetuximab resistance. Cetuximab-resistant clones of non-small cell lung cancer in culture and patient-derived xenografts in mice had increased abundance of AXL and nuclear EGFR (nEGFR). Cellular fractionation analysis, super-resolution microscopy, and electron microscopy revealed that genetic loss of AXL reduced the accumulation of nEGFR. SRC family kinases (SFKs) and HER family ligands promote the nuclear translocation of EGFR. We found that AXL knockdown reduced the expression of the genes encoding the SFK family members YES and LYN and the ligand neuregulin-1 (NRG1). AXL knockdown also decreased the interaction between EGFR and the related receptor HER3 and accumulation of HER3 in the nucleus. Overexpression of LYN and NRG1 in cells depleted of AXL resulted in accumulation of nEGFR, rescuing the deficit induced by lack of AXL. Collectively, these data uncover a previously unrecognized role for AXL in regulating the nuclear translocation of EGFR and suggest that AXL-mediated SFK and NRG1 expression promote this process.

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 (CtxR cells). Using RNAi methods and novel AXL-targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation, and MAPK signaling in CtxR cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in CtxR 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.

Triple-negative breast cancer (TNBC) is a subclass of breast cancers (i.e., estrogen receptor–negative, progesterone receptor–negative, and HER2-negative) that have poor prognosis and very few identified molecular targets. Strikingly, a high percentage of TNBCs overexpresses the EGF receptor (EGFR), yet EGFR inhibition has yielded little clinical benefit. Over the last decade, advances in EGFR biology have established that EGFR functions in two distinct signaling pathways: (i) classical membrane-bound signaling and (ii) nuclear signaling. Previous studies have demonstrated that nuclear EGFR (nEGFR) can enhance resistance to anti-EGFR therapies and is correlated with poor overall survival in breast cancer. On the basis of these findings, we hypothesized that nEGFR may promote intrinsic resistance to cetuximab in TNBC. To examine this question, a battery of TNBC cell lines and human tumors were screened and found to express nEGFR. Knockdown of EGFR expression demonstrated that TNBC cell lines retained dependency on EGFR for proliferation, yet all cell lines were resistant to cetuximab. Furthermore, Src Family Kinases (SFKs) influenced nEGFR translocation in TNBC cell lines and in vivo tumor models, where inhibition of SFK activity led to potent reductions in nEGFR expression. Inhibition of nEGFR translocation led to a subsequent accumulation of EGFR on the plasma membrane, which greatly enhanced sensitivity of TNBC cells to cetuximab. Collectively, these data suggest that targeting both the nEGFR signaling pathway, through the inhibition of its nuclear transport, and the classical EGFR signaling pathway with cetuximab may be a viable approach for the treatment of patients with TNBC. Mol Cancer Ther; 13(5); 1356–68. ©2014 AACR.

The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for use in oncology. Despite clinical success the majority of patients do not respond to cetuximab and those who initially respond frequently acquire resistance. To understand how tumor cells acquire resistance to cetuximab we developed a model of resistance using the non-small cell lung cancer line NCI-H226. We found that cetuximab-resistant (Ctx (R) ) clones manifested strong activation of EGFR, PI3K/AKT and MAPK. To investigate the role of AKT signaling in cetuximab resistance we analyzed the activation of the AKT pathway effector molecules using a human AKT phospho-antibody array. Strong activation was observed in Ctx (R) clones for several key AKT substrates including c-jun, GSK3β, eIF4E, rpS6, IKKα, IRS-1 and Raf1. Inhibition of AKT signaling by siAKT1/2 or by the allosteric AKT inhibitor MK-2206 resulted in robust inhibition of cell proliferation in all Ctx (R) clones. Moreover, the combinational treatment of cetuximab and MK-2206 resulted in further decreases in proliferation than either drug alone. This combinatorial treatment resulted in decreased activity of both AKT and MAPK thus highlighting the importance of simultaneous pathway inhibition to maximally affect the growth of Ctx (R) cells. Collectively, our findings demonstrate that AKT activation is an important pathway in acquired resistance to cetuximab and suggests that combinatorial therapy directed at both the AKT and EGFR/MAPK pathways may be beneficial in this setting.

The treatment of metastatic colorectal cancer (mCRC) remains one of the largest hurdles in cancer therapeutics to date. The most advanced treatment option for mCRC patients are anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) that bind to and inhibit the activity of EGFR. While the use of anti-EGFR mABs has had great impact in the treatment of mCRC, it has now been widely accepted that mCRC tumors with a mutation in the small GTPase KRAS do not respond to these therapies. KRAS mutations allow for EGFR independent activation of various oncogenic signaling cascades. In attempts to inhibit KRAS mutant tumor growth, BRAF, MEK and farsenyltransferase inhibitors have been used, however, their clinical efficacy is still accruing in the setting of CRC. Recent data suggests that various other inhibitors, including inhibitors of Src family kinases (SFK) and hepatocyte growth factor receptor (MET), may have potential preclinical and clinical success in KRAS mutant tumors. Additionally, it is becoming increasingly clear that different KRAS missense mutations may have varied biological responses to cetuximab, suggesting that cetuximab may still be a potential therapeutic option in some KRAS mutant tumors. In this review, we highlight the importance for both improved multimodality approaches for treating KRAS mutant mCRC tumors and stratification of KRAS mutations in response to different treatment regimes in order to optimize the best possible care for mCRC patients.

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.

Purpose: Head and neck squamous cell carcinoma (HNSCC) represents the eighth most common malignancy worldwide. Standard-of-care treatments for patients with HNSCC include surgery, radiation, and chemotherapy. In addition, the anti-EGFR monoclonal antibody cetuximab is often used in combination with these treatment modalities. Despite clinical success with these therapeutics, HNSCC remains a difficult malignancy to treat. Thus, identification of new molecular targets is critical. Experimental Design: In the current study, the receptor tyrosine kinase AXL was investigated as a molecular target in HNSCC using established cell lines, HNSCC patient-derived xenografts (PDX), and human tumors. HNSCC dependency on AXL was evaluated with both anti-AXL siRNAs and the small-molecule AXL inhibitor R428. Furthermore, AXL inhibition was evaluated with standard-of-care treatment regimens used in HNSCC. Results: AXL was found to be highly overexpressed in several models of HNSCC, where AXL was significantly associated with higher pathologic grade, presence of distant metastases, and shorter relapse-free survival in patients with HNSCC. Further investigations indicated that HNSCC cells were reliant on AXL for cellular proliferation, migration, and invasion. In addition, targeting AXL increased HNSCC cell line sensitivity to chemotherapy, cetuximab, and radiation. Moreover, radiation-resistant HNSCC cell line xenografts and PDXs expressed elevated levels of both total and activated AXL, indicating a role for AXL in radiation resistance. Conclusions: This study provides evidence for the role of AXL in HNSCC pathogenesis and supports further preclinical and clinical evaluation of anti-AXL therapeutics for the treatment of patients with HNSCC. Clin Cancer Res; 21(11); 2601–12. ©2015 AACR.

Background: Nearly 600,000 people are diagnosed with head and neck cancer worldwide and 60% succumb to the disease within 5 years. The epidermal growth factor receptor (EGFR) is a major driver of HNSCC and in 2006 the EGFR monoclonal antibody cetuximab was FDA-approved for HNSCC treatment. However, cetuximab has not conferred significant long-term benefit compared to chemoradiation in patients with poorly differentiated recurrent-metastatic HNSCC. We and others have demonstrated that cetuximab treatment activates alternative receptor tyrosine kinases (RTKs) including Fibroblast Growth Factor (FGF) receptors, Met and Axl in HNSCC. To circumvent administration of multiple RTK inhibitors in combination with cetuximab, we sought to identify a common molecular target that regulates the expression of these RTKs. Bromodomain-containing protein-4 (BRD4) has been shown to regulate the transcription of RTKs in breast cancer models. Hypothesis: We hypothesized that targeting BRD4 will overcome cetuximab resistance by depleting the expression of alternative RTKs in HNSCC. We further hypothesize that genetic and pharmacological targeting of BRD4 will synergize with cetuximab. Results: Using a phospho-RTK array, 72-hour cetuximab treatment increased or sustained phosphorylated levels of EGFR, HER2, HER3, MET, and AXL in several HNSCC cell lines. Treatment with the BRD4 inhibitor JQ1 abrogated both phosphorylated and total RTK abundance in the presence of cetuximab. Cetuximab and JQ1 robustly decreased phosphorylated Src and induced the senescence marker p21. Phenotypically, cetuximab and JQ1 significantly decreased survival and increased apoptosis in 6 HNSCC cell line models, while the normal oral keratinocyte cell line NOKsi had approximately 10-fold higher IC50s for the BRD4 inhibitors, JQ1 and I-BET762, relative to HNSCC cell lines. Importantly, two HNSCC models of acquired cetuximab resistance exhibited robust sensitivity to pharmacological (JQ1, IBET-762) and genetic (RNAi) BRD4 targeting strategies. Moreover, exogneous overexpression of different RTKs (HER3, ALK, and ROR1) resulted in cetuximab resistance that was reversed upon BRD4 targeting (RNAi and JQ1). Combination of cetuximab and JQ1 in co-culture experiments with T cells decreased the CD4+/CD25+ Treg population and PD-L1 expression on HNSCC cell lines. Further, preliminary findings indicate that JQ1 treatment prevents outgrowth of cetuximab-treated HNSCC patient-derived xenograft models. Conclusion: Our findings indicate that targeting BRD4 decreases the activation and expression of multiple RTKs that mediate resistance to the FDA-approved EGFR inhibitor cetuximab. Furthermore, BRD4 abrogates expression of immunosuppressive markers, making it a promising tumor intrinsic and extrinsic therapeutic strategy for HNSCC. Citation Format: Toni Brand, Yan Zeng, Brandon Leonard, Rachel O' Keefe, Hua Li, Daniel Johnson, Jennifer Grandis, Neil E. Bhola. Targeting BRD4 overcomes cetuximab resistance in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 95. doi:10.1158/1538-7445.AM2017-95

Radiation, cisplatin, and cetuximab are clinical therapeutics used in the treatment of head and neck squamous cell carcinoma (HNSCC). Despite clinical success with these modalities, development of both intrinsic and acquired resistance is an emerging problem in the management of HNSCC. Thus, identifying and targeting molecules driving this drug resistance is essential for improving efficacy of treatment approaches. Recent studies have identified a role for the TAM family of receptor tyrosine kinases (Tyro, Axl, and Mer) in tumor biology, especially the Axl receptor in promoting tumor growth and metastasis. Previously, we identified Axl as a logical molecular target in HNSCC and indicated that it may play a pivotal role in resistance to radiation, cisplatin, and cetuximab. In the current study, we advanced these early findings into pre-clinical models using patient-derived xenografts (PDXs). We have shown that small molecules targeting Axl can enhance therapy in PDXs that express high levels of Axl and have been identified as resistant to radiation, cisplatin, and cetuximab. Current studies are focusing on antibody-based targeting of Axl using PDXs and genetically-modified models of resistance. Furthermore, studies focused on co-targeting of Axl and Mer in vitro and in vivo have shown striking results and denote the importance of establishing logical approaches to target TAMs in the management of HNSCC. Finally, investigations into the molecular mechanisms of how Axl signaling can lead to resistance have underscored the importance of tyrosine 821 (Y821) of Axl. Overexpression of Axl rendered cetuximab sensitive lines resistant, but cell lines overexpressing Axl-Y821F retained their sensitivity to cetuximab. Advancing this line of study in vivo indicated that tumors expressing Axl were resistant to cetuximab whereas tumors harboring the Y821 mutation were sensitive demonstrating that signals emanating from Y821 may be critical for cetuximab resistant pathways. Collectively, the studies presented herein identify the TAM family of receptors as key players in radiation, cisplatin, and cetuximab resistance. These results provide rationale for the clinical targeting of TAM receptors to enhance the therapeutic modalities used in treating HNSCC. Citation Format: Nellie K. Black, Mari Iida, Tamara S. Rodems, Toni M. Brand, Randall J. Kimple, Deric L. Wheeler. Targeting TAM family members with antibody or small molecule inhibitors enhances therapeutic modalities of HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4176. doi:10.1158/1538-7445.AM2017-4176

Head and neck squamous cell carcinoma (HNSCC), the sixth most common cancer worldwide, has a five-year survival rate of only 50%. The first targeted agent FDA approved for treatment of HNSCC was cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR). Despite EGFR overexpression in up to 90% of HNSCC tumors and ample evidence supporting EGFR as a therapeutic target in HNSCC, the response rate for single-agent cetuximab is below 20%, and resistance to cetuximab-containing therapy remains a major obstacle in the effective treatment of HNSCC. Identification and targeting of mediators of cetuximab resistance is needed to improve patient outcomes. Secretion of the cytokine interleukin 6 (IL-6) has been proposed as a mechanism of resistance to cetuximab in HNSCC, and inhibition of signal transducer and activator of transcription 3 (STAT3), a downstream mediator of IL-6 signaling, has been shown to overcome cetuximab resistance in preclinical HNSCC models. Thus, we hypothesize that IL-6 signaling mediates resistance to cetuximab and that co-treatment with agents targeting the IL-6 pathway will enhance the therapeutic efficacy of cetuximab in HNSCC. We have generated cetuximab-resistant variants of the cetuximab-sensitive HNSCC cell lines Cal33 and PE/CA-PJ49. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis indicated that IL6 expression is increased 3.5- to 8-fold in cetuximab-resistant variants compared to the parental cells from which they were derived. In addition, a colony formation assay revealed that, as expected, cetuximab-treated parental cells formed fewer colonies than vehicle-treated cells, while cetuximab treatment had no effect on the ability of cetuximab-resistant PE/CA-PJ49 variants to form colonies. In contrast, treatment with tocilizumab, an IL-6 receptor-neutralizing monoclonal antibody, markedly reduced the colony-forming ability of the cetuximab-resistant variants, but not parental cells. These results suggest that targeted inhibition of IL-6 signaling may effectively inhibit proliferation in cetuximab-refractory HNSCC cells. Citation Format: Rachel A. O'Keefe, Neil Bhola, Toni M. Brand, Yan Zeng, Daniel E. Johnson, Jennifer R. Grandis. Targeting IL-6 signaling overcomes cetuximab resistance in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4108. doi:10.1158/1538-7445.AM2017-4108

NSCLC is a deadly disease that is driven by a multitude of factors. One of these factors is the epidermal growth factor receptor (EGFR). One of the most prominent molecular targeting agents to the EGFR is the antibody cetuximab. However, most patients develop resistance to this antibody. We have found in models of cetuximab resistance that the EGFR changes its location, to the nucleus, where it is not accessible to the large antibody. Our work over the last several years has discovered how to target the nEGFR, by blocking its translocation to the nucleus through Src Family Kinase blockade. In this first year we have determined 1) that nEGFR can serve as a prognostic factor in early stage NSCLC patients., 2) we have determined that we can target nEGFR in vivo and redistribute to the membrane in vivo, a critical first step for re-sensitizing to cetuximab and 3) developed a new avenue by developing a novel EGFR mutant that lacks its transcriptional potential. This will allow us to directly test the role of nEGFR in biology and cetuximab resistance.

The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR antibody that has been approved for use in oncology. Previously we investigated mechanisms of resistance to cetuximab using a model derived from the non-small cell lung cancer line NCI-H226. We demonstrated that cetuximab-resistant clones (Ctx(R)) had increased nuclear localization of the EGFR. This process was mediated by Src family kinases (SFKs), and nuclear EGFR had a role in resistance to cetuximab. To better understand SFK-mediated nuclear translocation of EGFR, we investigated which SFK member(s) controlled this process as well as the EGFR tyrosine residues that are involved. Analyses of mRNA and protein expression indicated upregulation of the SFK members Yes (v-Yes-1 yamaguchi sarcoma viral oncogene) and Lyn (v-yes-1 Yamaguchi sarcoma viral-related oncogene homolog) in all Ctx(R) clones. Further, immunoprecipitation analysis revealed that EGFR interacts with Yes and Lyn in Ctx(R) clones, but not in cetuximab-sensitive (Ctx(S)) parental cells. Using RNAi interference, we found that knockdown of either Yes or Lyn led to loss of EGFR translocation to the nucleus. Conversely, overexpression of Yes or Lyn in low nuclear EGFR-expressing Ctx(S) parental cells led to increased nuclear EGFR. Chromatin immunoprecipitation (ChIP) assays confirmed nuclear EGFR complexes associated with the promoter of the known EGFR target genes B-Myb and iNOS. Further, all Ctx(R) clones exhibited upregulation of B-Myb and iNOS at the mRNA and protein levels. siRNAs directed at Yes or Lyn led to decreased binding of EGFR complexes to the B-Myb and iNOS promoters based on ChIP analyses. SFKs have been shown to phosphorylate EGFR on tyrosines 845 and 1101 (Y845 and Y1101), and mutation of Y1101, but not Y845, impaired nuclear entry of the EGFR. Taken together, our findings demonstrate that Yes and Lyn phosphorylate EGFR at Y1101, which influences EGFR nuclear translocation in this model of cetuximab resistance.

Cetuximab is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody that has shown efficacy in metastatic colorectal cancer, head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC). Clinical data suggests that patients whom initially respond to cetuximab eventually acquire resistance, highlighting the importance of understanding resistant mechanisms for the development of better treatment regimes for cancer patients. Recent studies have identified a role for the TAM family of receptor tyrosine kinases (Tyro, Axl, and Mer) in tumor biology, and more specifically the Axl receptor as playing a key role in promoting tumor growth and metastasis. In the current study, we utilized NSCLC and HNSCC models of acquired resistance to cetuximab and found that Axl was overexpressed, activated, and highly associated with EGFR as compared parental controls. Further, using siRNA technology and novel Axl targeting agents (antibody and tyrosine kinase inhibitor), Axl was found to regulate proliferation, EGFR activation, and MAPK signaling in cetuximab resistant clones (CtxR). Additionally, Axl mRNA expression was regulated by EGFR induced MAPK activation in resistant cells. To investigate the role of Axl in in vivo models of acquired resistance to cetuximab, de novo acquired resistance was created by prolonged treatment of cetuximab sensitive (CtxS) tumor xenografts with cetuximab. Analysis of resultant CtxR tumors demonstrated that Axl was hyperactivated and highly associated with EGFR as compared to vehicle treated tumors. In addition, CtxR tumors expressing the highest levels of phosphorylated Axl also expressed hyperactivated EGFR, supporting the role of Axl in EGFR activation. Finally, to expand these findings to other members of the TAM family, Tyro, Axl, or Mer were stably overexpressed in CtxS parental NSCLC cells and tested for cetuximab response. The results of this experimentation indicated that stable overexpression of each TAM receptor rendered CtxS cells resistant to cetuximab therapy. Collectively, the studies presented herein identify the TAM family of receptors as key players in acquired resistance to cetuximab and provide rationale for the clinical evaluation of anti-TAM receptor therapeutics in the resistant setting. Citation Format: Toni Michel Brand, Mari Iida, Kelsey L. Corrigan, Neha Luthar, Megan Hornung, Mahmoud Toulany, Parkash Gill, Ravi Salgia, Deric L. Wheeler. The TAM family of receptor tyrosine kinases play a role in acquired resistance to cetuximab. [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 LB-215. doi:10.1158/1538-7445.AM2014-LB-215

Triple negative breast cancer (TNBC) is a subclass of breast cancer (i.e. estrogen receptor negative, progesterone receptor negative, and HER-2 negative) that has poor prognosis and very few identified molecular targets. Strikingly the majority of TNBC's overexpress the epidermal growth factor receptor (EGFR), yet EGFR inhibition has had very little clinical benefit in this setting. This suggests a gap in our knowledge of EGFR function in TNBC. Over the last decade, advances in the basic biology of EGFR in human cancer have established that the EGFR functions in two distinct signaling pathways: 1) Classical membrane bound signaling (classical EGFR pathway) and 2) nuclear signaling (nEGFR pathway). In the nEGFR pathway, published data from our laboratory suggests that the EGFR is phosphorylated by Src family kinases (SFKs) at tyrosine 1101 (Y1101) and this phosphorylation is a necessary and early event for trafficking EGFR from the membrane to the nucleus. Nuclear EGFR has been correlated with poor overall survival in breast cancer and has been shown to enhance resistance to the EGFR targeted therapies cetuximab and gefitinib. In the current study we found that TNBC cell lines were: 1) dependent on EGFR expression for proliferation as indicated by RNAi techniques, 2) resistant to cetuximab therapy and 3) harbored high levels of nEGFR and SFK activity. Further, a TNBC tissue microarray indicated that a portion of human TNBCs express nEGFR. Using molecular approaches to investigate if nEGFR and SFKs play a role in resistance to cetuximab we demonstrate that the overexpression of constitutively active Src (ca-Src) enhanced the nuclear localization of EGFR, while the overexpression of a negative regulator of Src, src-like adaptor protein (SLAP) prevented EGFR nuclear localization. SLAP not only associated with the EGFR but also led to a decrease in its phosphorylation at Y1101. Further, stable overexpression of several SFK members (Yes, Lyn, Fyn, Hck, Blk, and Fgr) in the breast cancer cell line MCF-7 led to both the activation of EGFR at Y1101 and potent increases in EGFR nuclear localization, indicating that SFKs exhibit functional redundancy in their ability to influence the nuclear translocation of the EGFR. Treatment of various cetuximab resistant TNBC cell lines with the small molecule SFK inhibitor dasatinib resulted in: 1) loss of nuclear localized EGFR, 2) increased plasma-membrane levels of the EGFR and 3) statistically significant, increases in cetuximab sensitivity. Taken together, these findings suggest targeting nEGFR, by abrogating its translocation to the nucleus via SFK inhibition, increases plasma-membrane levels of EGFR where it is susceptible to targeting by cetuximab. The combinatorial therapy of cetuximab and dasatinib results in the simultaneous targeting of both the classical and nEGFR signaling pathways, and therefore may enhance the therapeutic response of nEGFR expressing TNBCs to cetuximab. Citation Format: Toni M. Brand, Mari Iida, Neha Luthar, Matthew Wleklinski, Kellie Kostopoulos, Deric L. Wheeler. Nuclear EGFR serves as a functional molecular target in triple-negative breast 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 LB-219. doi:10.1158/1538-7445.AM2013-LB-219

The epidermal growth factor receptor (EGFR) family consists of four members including HER1 (EGFR), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Collectively this family of receptor tyrosine kinases (RTKs) play critical roles in the etiology of several human cancers as well as a role in resistance to radiation therapy. In 2006, a study in head and neck squamous cell carcinoma (HNSCC) indicated that using the anti-EGFR antibody cetuximab in combination with radiation resulted in a 10% advantage of 3-year overall survival rate when compared to radiotherapy alone. This study highlighted the therapeutic utility of RTK blockade in combination with radiation. Studies investigating the role of HER3 in radiation response are limited. In the present study, we investigated whether or not HER3 blockade could enhance radiation therapy using the HER3 antibody U3-1287/AMG888. We screened a battery of cell lines from lung, HNSCC and colorectal tumor lines for HER3 expression. The results indicated that all 15 cell lines tested showed expression of HER3. In addition, U3-1287/AMG888 was able to block basal HER3 activity and radiation induced HER3 activation. Further, proliferation assays, using U3-1287/AMG888, indicated that HER3 blockade could block proliferation in SCC6, SCC1483, H226 cell lines, highlighting the importance of HER3 in these tumor cells. Clonogenic assays showed that U3-1287/AMG888 could sensitize these lines to radiation. Furthermore, γ-H2AX analyses, detected by immunofluorescence, lead to a statistically significant increase in apoptotic cells. Cell cycle analysis, 24 and 48 hours after U3-1287/AMG888 and radiation treatments resulted in a significant G1 and G2 cell cycle arrest. Annexin-V binding assays showed a significant increase in apoptosis in the U3-1287/AMG888 plus radiation group as compared to either agent alone. To determine if HER3 blockade using U3-1287/AMG888 could enhance radiation therapy in vivo we performed tumor growth delay experiments using SCC6, SCC1483 and H226 xenografts in nude mice. Mice were inoculated and treated with 1) IgG, 2) U3-1287/AMG888, 3) radiation or 4) the combination. The results of these experiments indicated that the combination of U3-1287/AMG888 and radiation had a strong impact on tumor growth in studies using single dose or fractionated dosing. Tumor analysis indicated that radiation treatment activated HER3 in vivo and U3-1287/AMG888 could abrogate this activation. Collectively our findings in vitro and in vivo suggest that U3-1287/AMG888 in combination with radiation has an impact on cell and tumor growth by impacting cell cycle progression, increasing apoptosis and increasing DNA damage. These findings suggest that HER3 may play a critical role in response to radiation therapy and blocking its activity may be of strong therapeutic benefit in human tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5726. doi:1538-7445.AM2012-5726

The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR antibody that has been approved by FDA for the treatment of patients with head and neck squamous cell carcinoma and metastatic colorectal cancer. However, increasing evidence suggests that the vast majority of patients do not respond to cetuximab and those who initially respond subsequently acquire resistance. To determine how tumor cells acquire resistance to cetuximab we developed models of acquired resistance using the non-small cell lung cancer line NCI-H226. During investigations into the molecular mechanisms of acquired resistance we found that cetuximab-resistant clones manifested strong activation of HER3 through transphosphorylation by the EGFR and constitutive PI(3)K/AKT activity and ultimate escape from cetuximab therapy. To determine the extent of AKT signaling in cetuximab-resistant clones, we analyzed the activation of the AKT pathway using a human AKT phospho-antibody array, which measured the activity of 137 proteins in the AKT pathway. Results showed strong activation of several key AKT substrates including GSK3β, eIF4E, S6, IKKα, IRS-1, Bad, β-catenin and Raf1 in cetuximab-resistant clones. To determine if cetuximab-resistant clones had dependence on AKT signaling we utilized RNAi technology to knockdown AKT expression. The results demonstrated that loss of AKT signaling in cetuximab-resistant clones resulted in decreased proliferative potential. To determine if AKT inhibition could be a therapeutic approach to overcome cetuximab resistance we investigated the AKT inhibitor MK-2206, a recently developed allosteric inhibitor, in this model of acquired resistance to cetuximab. We treated cetuximab-resistant clones with 2.5 μM of MK2206 and interrogated the AKT pathway using the human AKT phospho-antibody array. Results from this antibody array showed that MK-2206 inhibited multiple downstream AKT targets including GSK3β, eIF4E, S6, IKKα, IRS-1, β-catenin and Raf1. We next measured the effects of MK2206 on cellular proliferation and apoptosis. We found that cetuximab-resistant clones treated with MK-2206 showed robust inhibition of cell proliferation and increased apoptosis as compared to vehicle controls. Furthermore, the combination of cetuximab and MK-2206 resulted in further decreases in proliferation and increased apoptosis than either drug alone. Thus, MK-2206 treatment of cetuximab-resistant clones re-sensitized resistant clones to cetuximab. This combinatorial treatment resulted in decreased phospho-AKT and phospho-MAPK, highlighting the importance of these two pathways in cetuximab-resistance cells. These results indicate that dual targeting of the EGFR and AKT might produce greater impact than either drug alone. Collectively, our findings indicate that AKT activation is an important pathway in acquired resistance to cetuximab and suggest a rationale for clinical strategies that investigate combinatorial therapy directed at both the EGFR and AKT in patients with acquired resistance to cetuximab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A146.

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

Background Cetuximab, an anti-EGFR monoclonal antibody, is used to treat several cancers. However, many patients who initially respond to cetuximab acquire resistance. To examine mechanisms of acquired resistance, we developed a series of cetuximab-resistant (CtxR) clones derived from the cetuximab sensitive (CtxS) non-small cell lung cancer (NSCLC) cell line H226. Previous studies characterizing this model revealed that: 1) EGFR was robustly overexpressed in CtxR clones due to decreased EGFR ubiquitination and degradation and 2) CtxR clones expressed increased HER2 and HER3 activation resulting in constitutive activation of the PI3K/AKT signaling axis. These findings suggest that dual targeting HER family receptors would be highly beneficial in the CtxR setting. Results Since HER3 has been implicated in resistance to EGFR inhibitors, the efficacy of dually targeting both EGFR and HER3 in CtxR models was evaluated. First, EGFR and HER3 expression were knocked down with siRNAs. Compared to the CtxS parental cell line (HP), all CtxR clones exhibited robust decreases in cell proliferation upon dual knockdown. Analysis of CtxR clones indicated that neuregulin-1 was highly overexpressed compared to HP cells. Incubation of HP cells with neuregulin-1 rendered them resistant to cetuximab. Next, dual treatment of CtxR clones with cetuximab and the HER3 neutralizing monoclonal antibody (mAb) U3-1287 led to potent anti-proliferative effects. Blockade of EGFR with cetuximab resulted in inactivation of MAPK, while blockade of HER3 with U3-1287 resulted in the inactivation of AKT. Treatment with both mAbs resulted in knockdown of both signaling pathways simultaneously. HER2 was also strongly inactivated upon dual mAb therapy, suggesting that this treatment regimen can diminish signaling from three HER family receptors. De novo CtxR H226 mouse xenografts were established to determine if dual therapy could overcome acquired resistance to cetuximab in vivo. Tumors that had acquired resistance to cetuximab were significantly growth delayed upon dual treatment of U3-1287 and cetuximab compared to those continued on cetuximab only. Combinatorial-treated xenograft tumors expressed decreased Ki67 and increased cleaved caspase-3 levels compared to tumors treated with either monotherapy. Conclusions These studies demonstrate that dually targeting HER family receptors with antibody-based therapies can overcome acquired resistance to cetuximab.