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Head and neck cancer patients taking NSAIDs with PIK3CA tumor alterations demonstrate improved survival. Studies in relevant preclinical models implicate signaling via COX2-mediated production of PGE2 as an underlying mechanism for this survival benefit., PIK3CA is the most commonly altered oncogene in head and neck squamous cell carcinoma (HNSCC). We evaluated the impact of nonsteroidal anti-inflammatory drugs (NSAIDs) on survival in a PIK3CA-characterized cohort of 266 HNSCC patients and explored the mechanism in relevant preclinical models including patient-derived xenografts. Among subjects with PIK3CA mutations or amplification, regular NSAID use (≥6 mo) conferred markedly prolonged disease-specific survival (DSS; hazard ratio 0.23, P = 0.0032, 95% CI 0.09–0.62) and overall survival (OS; hazard ratio 0.31, P = 0.0043, 95% CI 0.14–0.69) compared with nonregular NSAID users. For PIK3CA-altered HNSCC, predicted 5-yr DSS was 72% for NSAID users and 25% for nonusers; predicted 5-yr OS was 78% for regular NSAID users and 45% for nonregular users. PIK3CA mutation predicted sensitivity to NSAIDs in preclinical models in association with increased systemic PGE2 production. These findings uncover a biologically plausible rationale to implement NSAID therapy in PIK3CA-altered HNSCC.

We have mapped a global network of virus–host protein interactions by purification of the complete set of human papillomavirus (HPV) proteins in multiple cell lines followed by mass spectrometry analysis. Integration of this map with tumor genome atlases shows that the virus targets human proteins frequently mutated in HPV− but not HPV+ cancers, providing a unique opportunity to identify novel oncogenic events phenocopied by HPV infection. For example, we find that the NRF2 transcriptional pathway, which protects against oxidative stress, is activated by interaction of the NRF2 regulator KEAP1 with the viral protein E1. We also demonstrate that the L2 HPV protein physically interacts with the RNF20/40 histone ubiquitination complex and promotes tumor cell invasion in an RNF20/40-dependent manner. This combined proteomic and genetic approach provides a systematic means to study the cellular mechanisms hijacked by virally induced cancers. Significance: In this study, we created a protein–protein interaction network between HPV and human proteins. An integrative analysis of this network and 800 tumor mutation profiles identifies multiple oncogenesis pathways promoted by HPV interactions that phenocopy recurrent mutations in cancer, yielding an expanded definition of HPV oncogenic roles. Cancer Discov; 8(11); 1474–89. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333

Human papillomavirus (HPV) type 16 is implicated in approximately 75% of head and neck squamous cell carcinomas (HNSCC) that arise in the oropharynx, where viral expression of the E6 and E7 oncoproteins promote cellular transformation, tumor growth, and maintenance. An important oncogenic signaling pathway activated by E6 and E7 is the PI3K pathway, a key driver of carcinogenesis. The PI3K pathway is also activated by mutation or amplification of PIK3CA in over half of HPV(+) HNSCC. In this study, we investigated the efficacy of PI3K-targeted therapies in HPV(+) HNSCC preclinical models and report that HPV(+) cell line- and patient-derived xenografts are resistant to PI3K inhibitors due to feedback signaling emanating from E6 and E7. Receptor tyrosine kinase profiling indicated that PI3K inhibition led to elevated expression of the HER3 receptor, which in turn increased the abundance of E6 and E7 to promote PI3K inhibitor resistance. Targeting HER3 with siRNA or the mAb CDX-3379 reduced E6 and E7 abundance and enhanced the efficacy of PI3K-targeted therapies. Together, these findings suggest that cross-talk between HER3 and HPV oncoproteins promotes resistance to PI3K inhibitors and that cotargeting HER3 and PI3K may be an effective therapeutic strategy in HPV(+) tumors.Significance: These findings suggest a new therapeutic combination that may improve outcomes in HPV(+) head and neck cancer patients. Cancer Res; 78(9); 2383–95. ©2018 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.

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 TNBC’s overexpress the epidermal growth factor 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: 1) classical membrane-bound signaling, and 2) 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. Based on 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. Further, 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 TNBC patients.

In Figure 4C, it was identified that the Histone H3 and α-Tubulin purification control blots for YES and LYN overexpressing cells were duplicated. The original Histone H3 control blot was found and confirmed the published results, however, the α-Tubulin control blot was not found. This error was determined to not impact the scientific findings of this figure. The authors regret this error.

In the original version of [this article][1] ([1][2]), three figures are incorrect. Fig. 6 uses the same image to represent “No Antibody” for UW-SCC36 and UW-SCC6; Fig. 2C includes bar graphs that exhibit irregularities, including an improperly scaled y -axis; and Fig. 2B contains duplicate

Cytoskeletal and focal adhesion abnormalities are observed in several types of cancer, including lung cancer. We have previously reported that paxillin (PXN) was mutated, amplified, and overexpressed in a significant number of lung cancer patient samples, that PXN protein was upregulated in more advanced stages of lung cancer compared with lower stages, and that the PXN gene was also amplified in some pre-neoplastic lung lesions. Among the mutations investigated, we previously found that PXN variant A127T in lung cancer cells enhanced cell proliferation and focal adhesion formation and colocalized with the anti-apoptotic protein B Cell Lymphoma 2 (BCL-2), which is known to localize to the mitochondria, among other sites. To further explore the effects of activating mutations of PXN on mitochondrial function, we cloned and expressed wild-type PXN and variants containing the most commonly occurring PXN mutations (P46S, P52L, G105D, A127T, P233L, T255I, D399N, E423K, P487L, and K506R) in a GFP-tagged vector using HEK-293 human embryonic kidney cells. Utilizing live-cell imaging to systematically study the effects of wild-type PXN vs. mutants, we created a model that recapitulates the salient features of the measured dynamics and conclude that compared with wild-type, some mutant clones confer enhanced focal adhesion and lamellipodia formation (A127T, P233L, and P487L) and some confer increased association with BCL-2, Dynamin-related Protein-1 (DRP-1), and Mitofusion-2 (MFN-2) proteins (P233L and D399N). Further, PXN mutants, through their interactions with BCL-2 and DRP-1, could regulate cisplatin drug resistance in human lung cancer cells. The data reported herein suggest that mutant PXN variants play a prominent role in mitochondrial dynamics with direct implications on lung cancer progression and hence, deserve further exploration as therapeutic targets.

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.

The TAM family of receptor tyrosine kinases (RTKs) has been discovered to play a predominant role in promoting the growth, survival, and metastatic spread of several tumor types. AXL and MERTK are two TAM family RTKs that are overexpressed in head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), and non-small cell lung cancer (NSCLC), malignancies that are highly metastatic and lethal. The AXL receptor is the most well-characterized TAM receptor and has been found to mediate resistance to both conventional and targeted cancer therapies. Since AXL is overexpressed in aggressive tumor types, cancer patients are currently being enrolled in clinical trials testing AXL inhibitors. In the current study, we analyzed the efficacy of AXL inhibitors—both small molecule and monoclonal antibody therapy—in HNSCC, TNBC, and NSCLC preclinical models. We observed limited efficacy of anti-AXL targeting strategies across these different models, which was attributed to the upregulation of MERTK. MERTK was robustly overexpressed in cell lines and patient-derived xenografts treated with AXL inhibitors. Inhibition of MERTK sensitized HNSCC, TNBC, and NSCLC preclinical models to AXL inhibitors, leading to a more potent blockade of downstream signaling, decreased expansion of tumor cells in culture, and reduced tumor growth in vivo. Furthermore, ectopic overexpression of MERTK in AXL inhibitor-sensitive models resulted in resistance to AXL-targeting strategies. These results suggest that cotargeting both AXL and MERTK may be highly beneficial in a variety of tumor types where both receptors are expressed and may therefore prolong antitumor effects and improve the survival of patients with lethal malignancies. Citation Format: Nellie K. McDaniel, Christopher T. Cummings, Toni M. Brand, Mari Iida, Justus Hulse, Hannah E. Pearson, Rachel A. Orbuch, Olivia J. Ondracek, Kurtis D. Davies, Parkash Gill, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Randall J. Kimple, Paul M. Harari, Deborah DeRyckere, Douglas K. Graham, Deric L. Wheeler. MERTK mediates intrinsic and adaptive resistance to AXL-targeting agents [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A140.

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.

Human papillomavirus (HPV) 16 plays an etiologic role in a growing subset of head and neck squamous cell carcinoma (HNSCC), where viral expression of the E6 and E7 oncoproteins is necessary for tumor growth and maintenance. Although patients with HPV(+) tumors have a more favorable prognosis, there are currently no HPV-selective therapies. Accumulating evidence indicates that HPV oncoproteins can activate the Phosphoinositol-3-Kinase (PI3K) pathway, which contributes to transformation. Furthermore, PI3K is genomically activated by PIK3CA mutation or amplification in a disproportionately high number of HPV(+) tumors as compared to HPV(-) tumors. Based on this knowledge, we investigated the efficacy of PI3K-targeted therapies in preclinical models of HPV(+) HNSCC. Our results indicate that HPV(+) preclinical models were less sensitive to the PI3K inhibitors BYL719, BKM120, and BEZ235 as compared with HPV(-) models. Sensitivity of HPV(+) cell lines to PI3K inhibitors was increased upon knockdown of the E6 and E7 oncoproteins. Reciprocally, overexpression of E6 and E7 in HPV(-) cells rendered them resistant to PI3K-targeted therapies. Proteomic analyses indicated that treatment of HPV(+) cell lines and patient-derived xenografts (PDXs) with the PI3Kα inhibitor BYL719 induced expression of the receptor tyrosine kinase HER3, as well as E6 and E7. HER3 was found to regulate the abundance of E6 and E7 in the HPV(+) models. Targeting HER3 with siRNAs or the monoclonal antibody, KTN3379, blocked the increase in E6 and E7 protein levels following BYL719 treatment, and enhanced the efficacy of PI3K inhibitors in HPV(+) cell line and PDX models. Taken together, these results suggest that crosstalk between HER3 and HPV16-E6E7 can limit the efficacy of PI3K inhibitors, and that co-targeting HER3 and PI3K may be an effective therapeutic strategy in HPV(+) tumors. This abstract is also being presented as Poster 67. Citation Format: Toni M. Brand, Stefan Hartmann, Neil E. Bhola, Hua Li, Yan Zeng, Rachel O'Keefe, Max V. Ranall, Sourav Bandyopadhyay, Margaret Soucheray, Danielle L. Swaney, Nevan Krogan, Carolyn Kemp, Umamaheswar Duvvuri, Daniel E. Johnson, Michelle A. Ozbun, Julie E. Bauman, Jennifer R. Grandis. HER3 crosstalk with HPV16-E6E7 is a feedback resistance mechanism to PI3K-targeted therapies in head and neck cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr PR04.

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

Background: PIK3CA is the most commonly altered oncogene in head and neck squamous cell carcinoma (HNSCC). Chronic non-steroidal anti-inflammatory (NSAID) use has been shown to reduce mortality in PIK3CA-mutated colorectal cancer through an unknown mechanism. In this retrospective cohort analysis, we evaluated the impact of NSAIDs on HNSCC survival, and sought a biologically plausible explanation for the observed effect. Methods: The PIK3CA status of tumor tissue was determined by FISH and next generation sequencing for 266 HNSCC patients treated at the University of Pittsburgh Cancer Institute. Cox joint proportional hazards models were used to generate multivariate projections of disease-specific (DSS) and overall survival (OS). Prostaglandin E2 (PGE2) secretion and NSAID sensitivity were then assessed in HNSCC cell lines with or without PIK3CA mutation. Results: Chronic NSAID use (≥2 days/week for ≥6 months) was associated with greater disease-specific (HR 0.24; 95% CI, 0.09 - 0.62; p = 0.0032) and overall survival (HR 0.31; 95% CI, 0.14 - 0.69; p = 0.0043) in patients whose tumors harbor PIK3CA mutation and/or amplification. Survival was unchanged by chronic NSAID use in patients with wild-type, unamplified PIK3CA. Chronic NSAID users with PIK3CA-altered tumors had an increase in estimated 5-year DSS from 25% to 72% and in OS from 45% to 78%. Benefit was independent of age, stage, and human papillomavirus (HPV) status; all of which were controlled for in the model. HNSCC cells with endogenous or engineered PIK3CA mutation secreted higher levels of PGE2 and were more sensitive to growth inhibition by NSAID treatment. Conclusions: Chronic NSAID use was associated with markedly improved survival in patients with PIK3CA-altered HNSCC, but not in those with wild-type, unamplified PIK3CA. Preclinical studies implicate the PI3K-COX-PGE2 signaling axis as a potential mediator of NSAID response. A prospective, randomized controlled trial of NSAIDs in patients with PIK3CA-altered HNSCC is warranted. Citation Format: Matthew Louis Hedberg, Noah Peyser, William Gooding, Hua Li, Toni Brand, Victor Olivas, Trever Bivona, Simion Choisea, Lin Wang, Jonas Johnson, Uma Duvvuri, Robert Ferris, Daniel Johnson, Patrick Ha, Julie Bauman, Jennifer Grandis. Chronic NSAID use increases survival in PIK3CA-altered 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 1779. doi:10.1158/1538-7445.AM2017-1779

The epidermal growth factor receptor (EGFR) is an important therapeutic target in patients with various cancers. Unfortunately, resistance to anti-EGFR therapeutics is common. Previous studies in our laboratory identified two mechanisms of resistance to the EGFR monoclonal antibody cetuximab: First, the nuclear translocation of EGFR bypasses the inhibitory effects of cetuximab; and second, the receptor tyrosine kinase AXL mediates cetuximab resistance by maintaining EGFR activation and downstream signaling. On the basis of these findings, we hypothesized that AXL could mediate the nuclear translocation of EGFR in the setting of cetuximab resistance. We found that NSCLC cetuximab-resistant clones and patient-derived xenografts (PDXs) had increased abundance of nuclear EGFR (nEGFR) and AXL. Cellular fractionation techniques, super resolution microscopy, and electron microcopy revealed that genetic loss of AXL diminished the nuclear translocation and accumulation of EGFR. Building on previous studies indicating that SRC family kinases (SFKs) and HER family ligands mediate the nuclear translocation of EGFR, we found that AXL knockdown down-regulated the expression of the SFKs YES and LYN, and the ligand neuregulin-1 (NRG1). Furthermore, AXL knockdown decreased the interaction between EGFR and HER3 and the nuclear abundance of HER3. Nuclear localization of EGFR could be rescued only upon simultaneous overexpression of Lyn and NRG1 in cells depleted 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 activation and NRG1 expression are necessary and sufficient to regulate this process. Citation Format: Toni M. Brand, Mari Iida, Kelsey L. Corrigan, Cara M. Braverman, John P. Coan, Bailey G. Flanigan, Andrew P. Stein, Ravi Salgia, Jana Rolff, Randall J. Kimple, Deric L. Wheeler. The receptor tyrosine kinase AXL mediates nuclear translocation of the epidermal growth factor receptor [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 3333. doi:10.1158/1538-7445.AM2017-3333

Small cell lung cancer (SCLC) is a devastating disease, and current therapies have not greatly improved the 5-year survival rates. Topoisomerase (Top) inhibition is a treatment modality for SCLC; however, the response is short lived. Consequently, our research has focused on improving SCLC therapeutics through the identification of novel targets. Previously, we identified MNNG HOS transforming gene (MET) to be overexpressed and functional in SCLC. Herein, we investigated the therapeutic potential of combinatorial targeting of MET using SU11274 and Top1 using 7-ethyl-10-hydroxycamptothecin (SN-38). MET and TOP1 gene copy numbers and protein expression were determined in 29 patients with limited (n = 11) and extensive (n = 18) disease. MET gene copy number was significantly increased (>6 copies) in extensive disease compared with limited disease (P = 0.015). Similar TOP1 gene copy numbers were detected in limited and extensive disease. Immunohistochemical staining revealed a significantly higher Top1 nuclear expression in extensive (0.93) versus limited (0.15) disease (P = 0.04). Interestingly, a significant positive correlation was detected between MET gene copy number and Top1 nuclear expression (r = 0.5). In vitro stimulation of H82 cells revealed hepatocyte growth factor (HGF)–induced nuclear colocalization of p-MET and Top1. Furthermore, activation of the HGF/MET axis enhanced Top1 activity, which was abrogated by SU11274. Combination of SN-38 with SU11274 dramatically decreased SCLC growth as compared with either drug alone. Collectively, these findings suggest that the combinatorial inhibition of MET and Top1 is a potentially efficacious treatment strategy for SCLC. Mol Cancer Ther; 13(3); 576–84. ©2013 AACR.

HER3 is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. In the present study, we investigated the capacity of the HER3 blocking antibody, U3-1287/AMG888, to modulate the in vitro and in vivo radiation response of human squamous cell carcinomas of the lung and head and neck. We screened a battery of cell lines from these tumors for HER3 expression and demonstrated that all cell lines screened exhibited expression of HER3. Importantly, U3-1287/AMG888 treatment could block both basal HER3 activity and radiation induced HER3 activation. Proliferation assays indicated that HER3 blockade could decrease the proliferation of both HNSCC cell line SCC6 and NSCLC cell line H226. Further, we demonstrated that U3-1287/AMG888 can sensitize cells to radiation in clonogenic survival assays, in addition to increasing DNA damage as detected via λ-H2AX immunofluo-rescence. To determine if U3-1287/AMG888 could enhance radiation sensitivity in vivo we performed tumor growth delay experiments using SCC6, SCC1483, and H226 xenografts. The results of these experiments indicated that the combination of U3-1287/AMG888 and radiation could decrease tumor growth in studies using single or fractionated doses of radiation. Analysis of HER3 expression in tumor samples indicated that radiation treatment activated HER3 in vivo and that U3-1287/AMG888 could abrogate this activation. Immunohistochemistry analysis of SCC6 tumors treated with both U3-1287/AMG888 and a single dose of radiation demonstrated that various cell survival and proliferation markers could be reduced. Collectively our findings suggest that U3-1287/AMG888 in combination with radiation has an impact on cell and tumor growth by increasing DNA damage and cell death. These findings suggest that HER3 may play an important role in response to radiation therapy and blocking its activity in combination with radiation may be of therapeutic benefit in human tumors.

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.

Nuclear localized HER family receptor tyrosine kinases (RTKs) have been observed in primary tumor specimens and cancer cell lines for nearly two decades. Inside the nucleus, HER family members (EGFR, HER2, and HER3) have been shown to function as co-transcriptional activators for various cancer-promoting genes. However, the regions of each receptor that confer transcriptional potential remain poorly defined. The current study aimed to map the putative transactivation domains (TADs) of the HER3 receptor. To accomplish this goal, various intracellular regions of HER3 were fused to the DNA binding domain of the yeast transcription factor Gal4 (Gal4DBD) and tested for their ability to transactivate Gal4 UAS-luciferase. Results from these analyses demonstrated that the C-terminal domain of HER3 (CTD, amino acids distal to the tyrosine kinase domain) contained potent transactivation potential. Next, nine HER3-CTD truncation mutants were constructed to map minimal regions of transactivation potential using the Gal4 UAS-luciferase based system. These analyses identified a bipartite region of 34 (B₁) and 27 (B₂) amino acids in length that conferred the majority of HER3's transactivation potential. Next, we identified full-length nuclear HER3 association and regulation of a 122 bp region of the cyclin D1 promoter. To understand how the B₁ and B₂ regions influenced the transcriptional functions of nuclear HER3, we performed cyclin D1 promoter-luciferase assays in which HER3 deleted of the B₁ and B₂ regions was severely hindered in regulating this promoter. Further, the overexpression of HER3 enhanced cyclin D1 mRNA expression, while HER3 deleted of its identified TADs was hindered at doing so. Thus, the ability for HER3 to function as a transcriptional co-activator may be dependent on specific C-terminal TADs.

Introduction Nuclear EGFR (nEGFR) has been identified in various human tumor tissues, including cancers of the breast, ovary, oropharynx, and esophagus, and has predicted poor patient outcomes. We sought to determine if protein expression of nEGFR is prognostic in early stage non-small cell lung cancer (NSCLC). Methods Resected stages I and II NSCLC specimens were evaluated for nEGFR protein expression using immunohistochemistry (IHC). Cases with at least one replicate core containing ≥5% of tumor cells demonstrating strong dot-like nucleolar EGFR expression were scored as nEGFR positive. Results Twenty-three (26.1% of the population) of 88 resected specimens stained positively for nEGFR. Nuclear EGFR protein expression was associated with higher disease stage (45.5% of stage II vs. 14.5% of stage I; p =0.023), histology (41.7% in squamous cell carcinoma vs. 17.1% in adenocarcinoma; p =0.028), shorter progression-free survival (PFS) (median PFS 8.7 months [95% CI 5.1–10.7 mo] for nEGFR positive vs. 14.5 months [95% CI 9.5–17.4 mo] for nEGFR negative; hazard ratio (HR) of 1.89 [95% CI 1.15–3.10]; p =0.011), and shorter overall survival (OS) (median OS 14.1 months [95% CI 10.3–22.7 mo] for nEGFR positive vs. 23.4 months [95% CI 20.1–29.4 mo] for nEGFR negative; HR of 1.83 [95% CI 1.12–2.99]; p =0.014). Conclusions Expression of nEGFR protein was associated with higher stage and squamous cell histology, and predicted shorter PFS and OS, in this patient cohort. Nuclear EGFR serves as a useful independent prognostic variable and as a potential therapeutic target in NSCLC.

Background: Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with a rising incidence of more than 600,000 new cases each year. Human papillomavirus (HPV)16 plays an etiologic role in a growing subset of HNSCCs, where viral expression of the E6 and E7 oncoproteins are necessary for tumor growth and maintenance. Although patients with HPV(+) tumors have a more favorable prognosis compared with HPV(-) patients, there are currently no HPV-selective therapies. Recent studies have identified differential receptor tyrosine kinase (RTK) profiles in HPV(+) vs. HPV(-) tumors. One such RTK, HER3, is overexpressed and highly associated with phosphoinositide 3-kinase (PI3K) in HPV(+) tumors. Methods: In the current study, HER3 was investigated as a molecular target in HPV(+) HNSCC cell lines and patient-derived xenografts (PDXs). HNSCC dependency on HER3 was evaluated with both anti-HER3 siRNAs and the clinically advanced anti-HER3 monoclonal antibody, KTN3379. Furthermore, the anti-growth effects of combinatorial HER3 and PI3K inhibition was examined in HPV(+) HNSCC models. Results: HER3 was overexpressed and activated in HPV(+) HNSCC models. Further investigation indicated that HPV(+) HNSCC cells were reliant on HER3 for cellular proliferation and PI3K pathway signaling, whereas little effect was observed in HPV(-) cell lines. Intriguingly, HPV oncoproteins directly regulated HER3, where knockdown of E6 and E7 resulted in loss of total HER3 protein expression and reduced PI3K pathway signaling (p-AKT, p-p70S6K, p-rpS6) in HPV(+) cell lines. Conversely, overexpression of E6 and E7 increased total HER3 protein expression (5-7 fold) and PI3K pathway signaling in HPV(-) cell lines. Since HER3 signals via the PI3K pathway to mediate cellular proliferation and survival, we sought to identify if HER3 can mediate response to PI3K pathway inhibitors. Stimulation of HPV(+) cell lines with neuregulin-1, the cognate ligand of HER3, resulted in complete resistance to the p110α inhibitor, BYL719, and prevented BYL719 induced inhibition of AKT. Neuregulin-1 stimulation of HPV(-) cell lines had no effect on their response to BYL719, and did not rescue AKT activation. Furthermore, treatment of HPV(+) cell lines with BYL719 resulted in enhanced HER3 expression and activation (20-50 fold), indicating that HER3 may mediate resistance to PI3K inhibitors in HPV(+) cells. Finally, dual targeting of HER3 and PI3K synergistically inhibited HPV(+) cell line proliferation (by 50-70%) and resulted in sustained knockdown of PI3K pathway signaling. While HPV(-) cell lines were responsive to PI3K inhibition, HER3 blockade did not provide additional benefit. HPV(+) PDX models are currently being evaluated for response to dual kinase inhibition. Conclusions: These studies identify HER3 as a potential therapeutic target, and provide a rationale for the clinical evaluation of combined HER3 and PI3K inhibition in HPV(+) HNSCCs. Citation Format: Toni Michel Brand, Stefan Hartmann, Neil Bhola, Noah D. Peyser, Hua Li, Yan Zeng, Max V. Randall, Sourav Bandyopadhyay, Jennifer R. Grandis. HER3 is a functional molecular target in HPV-associated head and neck cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1205.

Triple-negative breast cancer (TNBC) is a subclass of breast cancers that are negative for the estrogen receptor, the progesterone receptor, and HER2. Currently, TNBCs have poor prognosis and very few proven molecular targets. One promising target in TNBC has been the epidermal growth factor receptor (EGFR), as it is overexpressed in as many as 80% of TNBCs. Strikingly, however, antibody-based therapy directed against the EGFR (cetuximab) in TNBCs has had modest clinical impact. Over the last decade, advances in EGFR biology have established that the EGFR functions in two distinct signaling pathways: classical membrane-bound signaling and the nuclear EGFR signaling pathway. We previously demonstrated that EGFR nuclear translocation is dependent on Src Family Kinases (SFKs) and phosphorylation of tyrosine 1101 (Y1101) on the cytoplasmic tail of the EGFR. In these studies, translocation of the EGFR to the nucleus led to activation of the nuclear EGFR signaling pathway, which resulted in cetuximab resistance. Resistance could be reversed by simultaneously targeting both the nuclear EGFR signaling pathway and the classical EGFR signaling pathway through the inhibition of its nuclear transport (blockade of SFKs) and with cetuximab, respectively. Despite the identification of nuclear EGFR as a functional molecular target in TNBC, the role of Y1101 in EGFR translocation has yet to be identified. In this study, we seek to better understand the role of Y1101 in the nuclear translocation of EGFR in TNBC. Using a battery of TNBC cell lines, we have created isogenic pairs that overexpress either wild type EGFR (EGFR-WT) or EGFR with a mutant Y1101 (EGFR-Y110F). Using electron and super-resolution confocal microscopy as well as cellular fractionation, we have shown that EGFR-Y1101 undergoes normal endocytosis upon EGF stimulation in multiple cell lines, but unlike EGFR-WT, EGFR-Y1101F deposits around the perinuclear region. These data suggest that EGFR-Y1101F is able to undergo normal translocation to the perinuclear space, but that amino acid Y1101 is necessary for direct nuclear entry, potentially due to its interactions with nucleoporins. Current work is focused on the identification of nucleoporins with which EGFR-Y1101 may interact. In addition, continued work focuses on allelic mutation using CrisprCas9. Ultimately, the long-term goal of this project is to understand how Y1101 mediates EGFR nuclear translocation. With this information, we will then aim to develop allosteric inhibitors of Y1101 that may abrogate EGFR nuclear translocation and lead to a novel therapeutic which targets the nuclear EGFR signaling pathway in TNBC. Citation Format: Bailey G. Flanigan, Mari Iida, Toni M. Brand, Deric L. Wheeler. Understanding the role of tyrosine 1101 in the nuclear translocation of the epidermal growth factor receptor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 247.

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.

A high frequency of somatic mutations has been found in breast cancers within the gene encoding the catalytic p110α subunit of PI3K, PIK3CA. Using isogenic human breast epithelial cells, we have previously demonstrated that oncogenic PIK3CA “hotspot” mutations predict for response to the toxic effects of lithium. However, other somatic genetic alterations occur within this pathway in breast cancers, and it is possible that these changes may also predict for lithium sensitivity. We overexpressed the epidermal growth factor receptor (EGFR) into the non-tumorigenic human breast epithelial cell line MCF-10A, and compared these cells to isogenic cell lines previously created via somatic cell gene targeting to model Pten loss, PIK3CA mutations, and the invariant AKT1 mutation, E17K. EGFR overexpressing clones were capable of cellular proliferation in the absence of EGF and were sensitive to lithium similar to the results previously seen with cells harboring PIK3CA mutations. In contrast, AKT1 E17K cells and PTEN−/− cells displayed resistance or partial sensitivity to lithium, respectively. Western blot analysis demonstrated that lithium sensitivity correlated with significant decreases in both PI3K and MAPK signaling that were observed only in EGFR overexpressing and mutant PIK3CA cell lines. These studies demonstrate that EGFR overexpression and PIK3CA mutations are predictors of response to lithium, whereas Pten loss and AKT1 E17K mutations do not predict for lithium sensitivity. Our findings may have important implications for the use of these genetic lesions in breast cancer patients as predictive markers of response to emerging PI3K pathway inhibitors.

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

e22065 Background: A large number of esophageal cancer patients present with advanced or metastatic disease and their overall prognosis is poor. In order to improve survival, we need to identify mo...

The HER family of receptor tyrosine kinases consist of four receptors, epidermal growth factor receptor (EGFR/ErbB1/HER1), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Collectively, this family of receptors plays a critical role in initiating proliferation and survival signals in several human cancers. It is well established that the HER family receptors rely on two distinct compartments of signaling: 1) Classical membrane bound signaling and 2) nuclear signaling. In the nucleus, HER family receptors can serve as co-transcription factors, mediated by their C-terminal domains, to promote transcription of several genes essential for cell proliferation and cell cycle regulation, including regulation of the gene cyclin D1. However, the domains within the C-terminus of EGFR, HER2 and HER3 that confer this transcriptional potential (transactivation domains) have yet to be defined. In the current study we aimed to minimally map the regions of the C-terminal domains of EGFR, HER2 and HER3 that function as transactivation domains. We first demonstrate that EGFR, HER2, and HER3 are nuclear localized in their full-length forms in various breast and lung cancer cell lines. Next, we fused various intracellular cytoplasmic domain regions of each receptor to the DNA binding domain of the yeast transcription factor Gal4, and measured the ability for each construct to transactivate Gal4 UAS-luciferase activity. This analysis demonstrated that the C-terminal region distal to the tyrosine kinase domain (CTD) of all HER family receptors have strong transactivation potential, with HER2 exhibiting the highest transactivation potential. Further deletion mapping analysis of each receptor's CTD identified two regions (bipartite) of approximately 25-40 amino acids in length that harbored the majority of the receptors transactivation potential. To understand how the identified bipartite C-terminal regions of each HER family receptor influenced their transcriptional functions we deleted these regions from each full-length receptor and performed cyclin D1 promoter-luciferase assays. While wild type EGFR, HER2, and HER3 overexpression was capable of transactivating cyclin D1-luciferase, bipartite deleted receptors were severely hindered in their ability to regulate the cyclin D1 promoter. Collectively, the findings presented herein suggest that the EGFR, HER2 and HER3 contain a bipartite C-terminal transactivation domain that may be responsible for their ability to function as co-transcription factors. Citation Format: Toni M. Brand, Mari Iida, Matthew J. Wleklinski, Neha Luthar, Megan M. Starr, Deric L. Wheeler. Mapping C-terminal transactivation domains of nuclear HER family receptor tyrosine kinases. [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 4276. doi:10.1158/1538-7445.AM2013-4276

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.