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Purpose: We previously reported that a fibroblast growth factor (FGF) receptor (FGFR) signaling pathway drives growth of lung cancer cell lines of squamous and large cell histologies. Herein, we explored FGFR dependency in cell lines derived from the tobacco-related malignancy, head and neck squamous cell carcinoma (HNSCC).Experimental Design: FGF and FGFR mRNA and protein expression was assessed in nine HNSCC cell lines. Dependence on secreted FGF2 for cell growth was tested with FP-1039, an FGFR1-Fc fusion protein. FGFR and epidermal growth factor receptor (EGFR) dependence was defined by sensitivity to multiple inhibitors selective for FGFRs or EGFR.Results: FGF2 was expressed in eight of the nine HNSCC cell lines examined. Also, FGFR2 and FGFR3 were frequently expressed, whereas only two lines expressed FGFR1. FP-1039 inhibited growth of HNSCC cell lines expressing FGF2, identifying FGF2 as an autocrine growth factor. FGFR inhibitors selectively reduced in vitro growth and extracellular signal-regulated kinase signaling in three HNSCC cell lines, whereas three distinct lines exhibited responsiveness to both EGFR and FGFR inhibitors. Combinations of these drugs yielded additive growth inhibition. Finally, three cell lines were highly sensitive to EGFR tyrosine kinase inhibitors (TKI) with no contribution from FGFR pathways.Conclusions: FGFR signaling was dominant or codominant with EGFR in six HNSCC lines, whereas three lines exhibited little or no role for FGFRs and were highly EGFR dependent. Thus, the HNSCC cell lines can be divided into subsets defined by sensitivity to EGFR and FGFR-specific TKIs. FGFR inhibitors may represent novel therapeutics to deploy alone or in combination with EGFR inhibitors in HNSCC. Clin Cancer Res; 17(15); 5016–25. ©2011 AACR.

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The FGFR1 is a therapeutic target under investigation in multiple solid tumors and clinical trials of selective tyrosine kinase inhibitors (TKI) are underway. Treatment with a single TKI represents a logical step toward personalized cancer therapy, but intrinsic and acquired resistance mechanisms limit their long-term benefit. In this study, we deployed RNAi-based functional genomic screens to identify protein kinases controlling the intrinsic sensitivity of FGFR1-dependent lung cancer and head and neck squamous cell cancer (HNSCC) cells to ponatinib, a multikinase FGFR-active inhibitor. We identified and validated a synthetic lethal interaction between MTOR and ponatinib in non–small cell lung carcinoma cells. In addition, treatment with MTOR-targeting shRNAs and pharmacologic inhibitors revealed that MTOR is an essential protein kinase in other FGFR1-expressing cancer cells. The combination of FGFR inhibitors and MTOR or AKT inhibitors resulted in synergistic growth suppression in vitro. Notably, tumor xenografts generated from FGFR1-dependent lung cancer cells exhibited only modest sensitivity to monotherapy with the FGFR-specific TKI, AZD4547, but when combined with the MTOR inhibitor, AZD2014, significantly attenuated tumor growth and prolonged survival. Our findings support the existence of a signaling network wherein FGFR1-driven ERK and activated MTOR/AKT represent distinct arms required to induce full transformation. Furthermore, they suggest that clinical efficacy of treatments for FGFR1-driven lung cancers and HNSCC may be achieved by combining MTOR inhibitors and FGFR-specific TKIs. Cancer Res; 75(20); 4398–406. ©2015 AACR.

Supplementary Methods, Figures S1-S12, Tables S1-S2. Figure S1. Overview of workflow for the synthetic lethal and essential gene screens. Figure S2. Procedure for isolating and barcoding PCR-amplified shRNA sequences. Figure S3. Growth inhibition of H157 cells by ponatinib in combination with MTOR inhibitor, AZD8055. Figure S4. Relative sensitivity of cancer cell lines to MTOR inhibitor AZD8055 using a cell proliferation assay. Figure S5. Dose response curves for growth inhibition of cancer cell lines by rapamycin. Figure S6. Growth inhibition by FGFR inhibitors in combination with MTOR inhibitor, AZD8055. Figure S7. Synergistic growth inhibition of CCL30 HNSCC cells by AZD4547 and AZD8055. Figure S8. Growth inhibition of Colo699 and H1581 cells by AZD4547 and rapamycin. Figure S9. Signal pathway inhibition by FGFR TKIs and rapamycin alone, and in combination. Figure S10. Growth inhibition by AKT inhibitors alone and combined with ponatinib on Colo699 cells. Figure S11. Combination ponatinib and AKT inhibitor GSK690693 yields synergistic in vitro growth inhibition. Figure S12. Combination AZD4547 and AZD2014 provides superior growth inhibition of Colo699 xenografts. Table S1. Primers used for sample preparation for Illumina sequencing. Table S2. Ranking of Synthetic Lethal Hits with ponatinib Treatment.

Targeted therapies have revolutionized cancer chemotherapy. Unfortunately, most patients develop multifocal resistance to these drugs within a matter of months. Here, we used a high-throughput phenotypic small molecule screen to identify MCB-613 as a compound that selectively targetsEGFR-mutant, EGFR inhibitor-resistant non-small cell lung cancer (NSCLC) cells harboring diverse resistance mechanisms. Subsequent proteomic and functional genomic screens involving MCB-613 identified its target in this context to be KEAP1, revealing that this gene is selectively essential in the setting of EGFR inhibitor resistance. In-depth molecular characterization demonstrated that (1) MCB-613 binds KEAP1 covalently; (2) a single molecule of MCB-613 is capable of bridging two KEAP1 monomers together; and, (3) this modification interferes with the degradation of canonical KEAP1 substrates such as NRF2. Surprisingly, NRF2 knockout sensitizes cells to MCB-613, suggesting that the drug functions through modulation of an alternative KEAP1 substrate. Together, these findings advance MCB-613 as a new tool for exploiting the selective essentiality of KEAP1 in drug-resistant,EGFR-mutant NSCLC cells.

Recent work in cell culture models, animal models, and human patients indicates that cancers with acquired resistance to a drug can become simultaneously dependent upon the presence of that drug for survival. This drug dependence offers a potential avenue for improving treatments aimed at slowing resistance, yet relatively little is known about the frequency with which drug dependence arises, the mechanisms underlying that dependence, and how drug schedules might be tuned to optimally exploit drug dependence. In this work, we address these open questions using a combination of laboratory evolution, in vitro experiments, and simple mathematical models. First, we used laboratory evolution to select more than 100 resistant BRAF mutant melanoma cell lines with acquired resistance to BRAF, MEK, or ERK inhibitors. We found that nearly half of these lines exhibit drug dependence, and the dependency response is associated with EGFR-driven senescence induction, but not apoptosis, following drug withdrawal. Then, using melanoma populations with evolved resistance to the BRAF inhibitor PLX4720, we showed that drug dependence can be leveraged to dramatically reduce population growth when treatment strategies include optimally chosen drug-free “holidays”. On short timescales, the duration of these holidays depends sensitively on the composition of the population, but for sufficiently long treatments it depends only on a single dimensionless parameter (γ) that describes how the growth rates of each cell type depend on the different treatment environments. Experiments confirm that the optimal holiday duration changes in time–with holidays of different durations leading to optimized treatments on different timescales. Furthermore, we find that the presence of “non-dependent” resistant cells does not change the optimal treatment schedule but leads to a net increase in population size. Finally, we show that even in the absence of detailed information about the composition and growth characteristics of cellular clones within a population, a simple adaptive therapy protocol can produce near-optimal outcomes using only measurements of total population size, at least when these measurements are sufficiently frequent. As a whole, these results may provide a stepping-stone toward the eventual development of evolution-inspired treatment strategies for drug dependent cancers.

Local adaptation directs populations towards environment-specific fitness maxima through acquisition of positively selected traits. However, rapid environmental changes can identify hidden fitness trade-offs that turn adaptation into maladaptation, resulting in evolutionary traps. Cancer, a disease that is prone to drug resistance, is in principle susceptible to such traps. We therefore performed pooled CRISPR-Cas9 knockout screens in acute myeloid leukemia (AML) cells treated with various chemotherapies to map the drug-dependent genetic basis of fitness trade-offs, a concept known as antagonistic pleiotropy (AP). We identified a PRC2-NSD2/3-mediated MYC regulatory axis as a drug-induced AP pathway whose ability to confer resistance to bromodomain inhibition and sensitivity to BCL-2 inhibition templates an evolutionary trap. Across diverse AML cell-line and patient-derived xenograft models, we find that acquisition of resistance to bromodomain inhibition through this pathway exposes coincident hypersensitivity to BCL-2 inhibition. Thus, drug-induced AP can be leveraged to design evolutionary traps that selectively target drug resistance in cancer.

Aerobic glycolysis or the Warburg effect (WE) is characterized by increased glucose uptake and incomplete oxidation to lactate. Although the WE is ubiquitous, its biological role remains controversial, and whether glucose metabolism is functionally different during fully oxidative glycolysis or during the WE is unknown. To investigate this question, here we evolved resistance to koningic acid (KA), a natural product that specifically inhibits glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a rate-controlling glycolytic enzyme, during the WE. We found that KA-resistant cells lose the WE but continue to conduct glycolysis and surprisingly remain dependent on glucose as a carbon source and also on central carbon metabolism. Consequently, this altered state of glycolysis led to differential metabolic activity and requirements, including emergent activities in and dependences on fatty acid metabolism. These findings reveal that aerobic glycolysis is a process functionally distinct from conventional glucose metabolism and leads to distinct metabolic requirements and biological functions.

The inhibitory epidermal growth factor receptor (EGFR) antibody, cetuximab, is an approved therapy for head and neck squamous cell carcinoma (HNSCC). Despite tumor response observed in some HNSCC patients, cetuximab alone or combined with radio- or chemotherapy fails to yield long-term control or cures. We hypothesize that a flexible receptor tyrosine kinase coactivation signaling network supports HNSCC survival in the setting of EGFR blockade, and that drugs disrupting this network will provide superior tumor control when combined with EGFR inhibitors. In this work, we submitted EGFR-dependent HNSCC cell lines to RNA interference-based functional genomics screens to identify, in an unbiased fashion, essential protein kinases for growth and survival as well as synthetic lethal targets for combined inhibition with EGFR antagonists. Mechanistic target of rapamycin kinase (MTOR) and erythroblastosis oncogene B (ERBB)3 were identified as high-ranking essential kinase hits in the HNSCC cell lines. MTOR dependency was confirmed by distinct short hairpin RNAs (shRNAs) and high sensitivity of the cell lines to AZD8055, whereas ERBB3 dependency was validated by shRNA-mediated silencing. Furthermore, a synthetic lethal kinome shRNA screen with a pan-ERBB inhibitor, AZD8931, identified multiple components of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway, consistent with ERK reactivation and/or incomplete ERK pathway inhibition in response to EGFR inhibitor monotherapy. As validation, distinct mitogen-activated protein kinase kinase (MEK) inhibitors yielded synergistic growth inhibition when combined with the EGFR inhibitors, gefitinib and AZD8931. The findings identify ERBB3 and MTOR as important pharmacological vulnerabilities in HNSCC and support combining MEK and EGFR inhibitors to enhance clinical efficacy in HNSCC. SIGNIFICANCE STATEMENT: Many cancers are driven by nonmutated receptor tyrosine kinase coactivation networks that defy full inhibition with single targeted drugs. This study identifies erythroblastosis oncogene B (ERBB)3 as an essential protein kinase in epidermal growth factor receptor–dependent head and neck squamous cell cancer (HNSCC) cell lines and a synthetic lethal interaction with the extracellular signal-regulated kinase mitogen-activated protein kinase pathway that provides a rationale for combining pan-ERBB and mitogen-activated protein kinase inhibitors as a therapeutic approach in subsets of HNSCC.

// Amanda B. Pilling 1,2 , Jihye Kim 1 , Adriana Estrada-Bernal 1 , Qiong Zhou 1 , Anh T. Le 1 , Katherine R. Singleton 1 , Lynn E. Heasley 1 , Aik Choon Tan 1 , James DeGregori 1 and Robert C. Doebele 1 1 University of Colorado Cancer Center, Aurora, CO, USA 2 Henry Ford Cancer Institute, Detroit, MI, USA Correspondence to: Robert C. Doebele, email: // Keywords : ALK; ROS1; NSCLC; MYC; synthetic lethality Received : July 28, 2017 Accepted : January 09, 2018 Published : January 16, 2018 Abstract A subset of lung cancers is dependent on the anaplastic lymphoma kinase ( ALK ) oncogene for survival, a mechanism that is exploited by the use of the ALK inhibitor crizotinib. Despite exceptional initial tumor responses to ALK inhibition by crizotinib, durable clinical response is limited and the emergence of drug resistance occurs. Furthermore, intrinsic resistance is frequently observed, where patients fail to respond initially to ALK-inhibitor therapy. These events demonstrate the underlying complexity of a molecularly-defined oncogene-driven cancer and highlights the need to identify compensating survival pathways. Using a loss-of-function whole genome short-hairpin (shRNA) screen, we identified MYCBP as a determinant of response to crizotinib, implicating the MYC signaling axis in resistance to crizotinib-treated ALK+ NSCLC. Further analysis reveals that ALK regulates transcriptional expression of MYC and activates c-MYC transactivation of c-MYC target genes. Inhibition of MYC by RNAi or small molecules sensitizes ALK+ cells to crizotinib. Taken together, our findings demonstrate a dual oncogene mechanism, where ALK positively regulates the MYC signaling axis, providing an additional oncogene target whose inhibition may prevent or overcome resistance.

Summary: Diverse pathways drive resistance to BRAF/MEK inhibitors in BRAF-mutant melanoma, suggesting that durable control of resistance will be a challenge. By combining statistical modeling of genomic data from matched pre-treatment and post-relapse patient tumors with functional interrogation of >20 in vitro and in vivo resistance models, we discovered that major pathways of resistance converge to activate the transcription factor, c-MYC (MYC). MYC expression and pathway gene signatures were suppressed following drug treatment, and then rebounded during progression. Critically, MYC activation was necessary and sufficient for resistance, and suppression of MYC activity using genetic approaches or BET bromodomain inhibition was sufficient to resensitize cells and delay BRAFi resistance. Finally, MYC-driven, BRAFi-resistant cells are hypersensitive to the inhibition of MYC synthetic lethal partners, including SRC family and c-KIT tyrosine kinases, as well as glucose, glutamine, and serine metabolic pathways. These insights enable the design of combination therapies that select against resistance evolution. : Diverse pathways drive resistance to BRAF/MEK inhibitors in BRAF-mutant melanoma, but by combining statistical modeling of tumor data with functional interrogation of resistance models, Singleton et al. show that these pathways converge to activate MYC. BRAFi-resistant cells are hypersensitive to the inhibition of MYC synthetic lethal partners, informing therapies that select against resistance. Keywords: melanoma, cancer therapeutics, therapeutic resistance, signaling networks, MYC, metabolism, synthetic lethality

SUMMARYAerobic glycolysis or the Warburg Effect (WE) is characterized by increased glucose uptake and incomplete oxidation to lactate. Although ubiquitous, the biological role of the WE remains controversial and whether glucose metabolism is functionally different during fully oxidative glycolysis or during the WE is unknown. To investigate this question, we evolved resistance to koningic acid (KA), a natural product shown to be a specific inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a rate-controlling glycolytic enzyme during the WE. We find that KA-resistant cells lose the WE but conduct glycolysis and surprisingly remain dependent on glucose and central carbon metabolism. Consequentially this altered state of glycolysis leads to differential metabolic activity and requirements including emergent activities in and dependencies on fatty acid metabolism. Together, these findings reveal that, contrary to some recent reports, aerobic glycolysis is a functionally distinct entity from conventional glucose metabolism and leads to distinct metabolic requirements and biological functions.

Diverse pathways can drive resistance to BRAF and MEK inhibitors in BRAF mutant melanoma, suggesting that durable control of resistance will be a major challenge. By combining statistical modeling of genomic data from matched pre-treatment and post-relapse patient tumors with functional interrogation of over 20 distinct in vitro and in vivo resistance models, we discovered that major pathways of resistance converge to activate the transcription factor c-MYC (MYC). MYC expression and pathway gene signatures were suppressed following initial drug treatment, then rebounded during tumor progression independent of the upstream pathway driving resistance. Critically, MYC activation was both necessary and sufficient for resistance, and consequently, suppression of MYC activity using either genetic approaches or BET bromodomain inhibition was sufficient to both resensitize diverse BRAFi-resistant models and delay resistance in treatment naive models. Finally, MYC-driven, BRAFi-resistant cells are hypersensitive to the inhibition of MYC synthetic lethal partners, including SRC family and c-KIT tyrosine kinases as well as glucose, glutamine, and serine metabolic pathways. Together, these insights enable the rational design of combination therapies that uniquely select against resistance evolution.

The clinical success of tyrosine kinase inhibitors specific for BCR-ABL-, EGFR-, ALK-, and ROS1-driven cancers continues to spur the quest to match specific oncogene-defined tumor types with an appropriate molecularly targeted therapy. Unfortunately, responses to these agents are not durable with intrinsic or acquired resistance limiting benefit. Additionally, efforts to identify the appropriate targets of new drugs have focused on nonfunctional assays such as large-scale sequencing for somatic mutations or analysis of gene copy number. Acknowledging both the problem of resistance and the shortcomings of the current methods for detecting appropriate drug targets, much interest has been focused on RNAi-based screens. These screens utilize a library of shRNAs targeting the whole genome or a subset of genes and provide a high-throughput and unbiased means to functionally assess genes impacting various aspects of tumor biology, especially proliferation and survival. The function of genes can be measured in the context of a specific drug treatment, termed a synthetic lethal screen, or genes may be assessed for their individual dependency, termed an essential gene screen. Here, we describe a method for performing both of these types of screens using a kinome-targeted shRNA library in human cancer cell lines.

Our laboratory has previously shown that some gefitinib-insensitive head and neck squamous cell carcinoma (HNSCC) cell lines exhibit dominant autocrine fibroblast growth factor receptor (FGFR) signaling. Herein, we deployed a whole-genome loss-of-function screen to identify genes whose knockdown potentiated the inhibitory effect of the FGFR inhibitor, AZ8010, in HNSCC cell lines. Three HNSCC cell lines expressing a genome-wide small hairpin RNA (shRNA) library were treated with AZ8010 and the abundance of shRNA sequences was assessed by deep sequencing. Under-represented shRNAs in treated cells are expected to target genes important for survival with AZ8010 treatment. Synthetic lethal hits were validated with specific inhibitors and independent shRNAs. We found that multiple alternate receptors provided protection from FGFR inhibition, including receptor tyrosine kinases (RTKs), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), and hepatocyte growth factor receptor (MET). We showed that specific knockdown of either ERBB2 or MET in combination with FGFR inhibition led to increased inhibition of growth relative to FGFR tyrosine kinase inhibitor (TKI) treatment alone. These results were confirmed using specific small molecule inhibitors of either ERBB family members or MET. Moreover, the triple combination of FGFR, MET, and ERBB family inhibitors showed the largest inhibition of growth and induction of apoptosis compared with the double combinations. These results reveal a role for alternate RTKs in maintaining progrowth and survival signaling in HNSCC cells in the setting of FGFR inhibition. Thus, improved therapies for HNSCC patients could involve rationally designed combinations of TKIs targeting FGFR, ERBB family members, and MET.

Functional genomics approaches such as gain- and loss-of-function screening can efficiently reveal genes that control cancer cell growth, survival, signal transduction, and drug resistance, but distilling the results of large-scale screens into actionable therapeutic strategies is challenging given our incomplete understanding of the functions of many genes. Research over several decades, including the results of large-scale cancer sequencing projects, has made it clear that many oncogenic properties are controlled by a common set of core oncogenic signaling pathways. By directly screening this core set of pathways, rather than much larger numbers of individual genes, it may be possible to more directly and efficiently connect functional genomic screening results with therapeutic targets. Here, we describe the recent development of methods to directly screen oncogenic pathways in high-throughput. We summarize the results of studies that have used pathway-centric screening to map the pathways of resistance to targeted therapies in diverse cancer types, then conclude by expanding on potential future applications of this approach.

Purpose: We previously reported that a fibroblast growth factor (FGF) receptor (FGFR) signaling pathway drives growth of lung cancer cell lines of squamous and large cell histologies. Herein, we explored FGFR dependency in cell lines derived from the tobacco-related malignancy, head and neck squamous cell carcinoma (HNSCC). Experimental Design: FGF and FGFR mRNA and protein expression was assessed in nine HNSCC cell lines. Dependence on secreted FGF2 for cell growth was tested with FP-1039, an FGFR1-Fc fusion protein. FGFR and epidermal growth factor receptor (EGFR) dependence was defined by sensitivity to multiple inhibitors selective for FGFRs or EGFR. Results: FGF2 was expressed in eight of the nine HNSCC cell lines examined. Also, FGFR2 and FGFR3 were frequently expressed, whereas only two lines expressed FGFR1. FP-1039 inhibited growth of HNSCC cell lines expressing FGF2, identifying FGF2 as an autocrine growth factor. FGFR inhibitors selectively reduced in vitro growth and extracellular signal-regulated kinase signaling in three HNSCC cell lines, whereas three distinct lines exhibited responsiveness to both EGFR and FGFR inhibitors. Combinations of these drugs yielded additive growth inhibition. Finally, three cell lines were highly sensitive to EGFR tyrosine kinase inhibitors (TKI) with no contribution from FGFR pathways. Conclusions: FGFR signaling was dominant or codominant with EGFR in six HNSCC lines, whereas three lines exhibited little or no role for FGFRs and were highly EGFR dependent. Thus, the HNSCC cell lines can be divided into subsets defined by sensitivity to EGFR and FGFR-specific TKIs. FGFR inhibitors may represent novel therapeutics to deploy alone or in combination with EGFR inhibitors in HNSCC. Clin Cancer Res; 17(15); 5016–25. ©2011 AACR.

The goal of this study was to determine if the high [K + ] in tears, 20–25 mM, serves to protect corneal epithelial cells from going into apoptosis after exposure to ambient UV-B radiation. Human corneal–limbal epithelial (HCLE) cells in culture were exposed to UV-B at doses of 50–200 mJ/cm 2 followed by measurement of K + channel activation and activity of apoptotic pathways. Patch-clamp recording showed activation of K + channels after UV-B exposure at 80 mJ/cm 2 or 150 mJ/cm 2 and a decrease in UV-induced K + efflux with increasing [K + ] o . The UV-activated current was partially blocked by the specific K + channel blocker, BDS-1. DNA fragmentation, as measured by the TUNEL assay, was induced after exposure to UV-B at 100–200 mJ/cm 2 . DNA fragmentation was significantly decreased when cells were incubated in 25, 50 or 100 mM K o + after exposure to UV-B. The effector caspase, caspase-3, was activated by exposure to UV-B at 50–200 mJ/cm 2 , but there was a significant decrease in activation when the cells were incubated in 25, 50 or 100 mM K o + following exposure to UV-B. A decrease in mitochondrial potential, a possible activator of caspase-3, occurred after exposure to UV-B at 100–200 mJ/cm 2 . This decrease in mitochondrial potential was prevented by 100 mM K o + ; however, 25 or 50 mM K o + provided minimal protection. Caspase-9, which is in the pathway from mitochondrial potential change to caspase-3 activation, showed little activation by UV-B radiation. Caspase-8, an initiator caspase that activates caspase-3, was activated by exposure to UV-B at 50–200 mJ/cm 2 , and this UV-activation was significantly reduced by 25–100 mM K o + . The data show that the physiologically relevant [K + ] o of 25 mM can inhibit UV-B induced activation of apoptotic pathways. This suggests that the relatively high [K + ] in tears reduces loss of K + from corneal epithelial cells in response to UV exposure, thereby contributing to the protection of the ocular surface from ambient UV radiation.

The epidermal growth factor receptor (EGFR) is overexpressed in approximately 90% of head and neck squamous cell carcinomas (HNSCC), and molecularly targeted therapy against the EGFR with the monoclonal antibody cetuximab modestly increases overall survival in head and neck cancer patients. We hypothesize that co-signaling through additional pathways limits the efficacy of cetuximab and EGFR-specific tyrosine kinase inhibitors (TKIs) in the clinical treatment of HNSCC. Analysis of gene expression changes in HNSCC cell lines treated 4 days with TKIs targeting EGFR and/or fibroblast growth factor receptors (FGFRs) identified transforming growth factor beta 2 (TGF-β2) induction in the three cell lines tested. Measurement of TGF-β2 mRNA validated this observation and extended it to additional cell lines. Moreover, TGF-β2 mRNA was increased in primary patient HNSCC xenografts treated for 4 weeks with cetuximab, demonstrating in vivo relevance of these findings. Functional genomics analyses with shRNA libraries identified TGF-β2 and TGF-β receptors (TGFβRs) as synthetic lethal genes in the context of TKI treatment. Further, direct RNAi-mediated silencing of TGF-β2 inhibited cell growth, both alone and in combination with TKIs. Also, a pharmacological TGFβRI inhibitor similarly inhibited basal growth and enhanced TKI efficacy. In summary, the studies support a TGF-β2-TGFβR pathway as a TKI-inducible growth pathway in HNSCC that limits efficacy of EGFR-specific inhibitors.

The FGFR1 is a therapeutic target under investigation in multiple solid tumors and clinical trials of selective tyrosine kinase inhibitors (TKI) are underway. Treatment with a single TKI represents a logical step toward personalized cancer therapy, but intrinsic and acquired resistance mechanisms limit their long-term benefit. In this study, we deployed RNAi-based functional genomic screens to identify protein kinases controlling the intrinsic sensitivity of FGFR1-dependent lung cancer and head and neck squamous cell cancer (HNSCC) cells to ponatinib, a multikinase FGFR-active inhibitor. We identified and validated a synthetic lethal interaction between MTOR and ponatinib in non–small cell lung carcinoma cells. In addition, treatment with MTOR-targeting shRNAs and pharmacologic inhibitors revealed that MTOR is an essential protein kinase in other FGFR1-expressing cancer cells. The combination of FGFR inhibitors and MTOR or AKT inhibitors resulted in synergistic growth suppression in vitro. Notably, tumor xenografts generated from FGFR1-dependent lung cancer cells exhibited only modest sensitivity to monotherapy with the FGFR-specific TKI, AZD4547, but when combined with the MTOR inhibitor, AZD2014, significantly attenuated tumor growth and prolonged survival. Our findings support the existence of a signaling network wherein FGFR1-driven ERK and activated MTOR/AKT represent distinct arms required to induce full transformation. Furthermore, they suggest that clinical efficacy of treatments for FGFR1-driven lung cancers and HNSCC may be achieved by combining MTOR inhibitors and FGFR-specific TKIs. Cancer Res; 75(20); 4398–406. ©2015 AACR.

Cancer cells can activate diverse signaling pathways to evade the cytotoxic action of drugs. We created and screened a library of barcoded pathway-activating mutant complementary DNAs to identify those that enhanced the survival of cancer cells in the presence of 13 clinically relevant, targeted therapies. We found that activation of the RAS-MAPK (mitogen-activated protein kinase), Notch1, PI3K (phosphoinositide 3-kinase)-mTOR (mechanistic target of rapamycin), and ER (estrogen receptor) signaling pathways often conferred resistance to this selection of drugs. Activation of the Notch1 pathway promoted acquired resistance to tamoxifen (an ER-targeted therapy) in serially passaged breast cancer xenografts in mice, and treating mice with a γ-secretase inhibitor to inhibit Notch signaling restored tamoxifen sensitivity. Markers of Notch1 activity in tumor tissue correlated with resistance to tamoxifen in breast cancer patients. Similarly, activation of Notch1 signaling promoted acquired resistance to MAPK inhibitors in BRAF(V600E) melanoma cells in culture, and the abundance of Notch1 pathway markers was increased in tumors from a subset of melanoma patients. Thus, Notch1 signaling may be a therapeutic target in some drug-resistant breast cancers and melanomas. Additionally, multiple resistance pathways were activated in melanoma cell lines with intrinsic resistance to MAPK inhibitors, and simultaneous inhibition of these pathways synergistically induced drug sensitivity. These data illustrate the potential for systematic identification of the signaling pathways controlling drug resistance that could inform clinical strategies and drug development for multiple types of cancer. This approach may also be used to advance clinical options in other disease contexts.

Worldwide, head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer. Because the epidermal growth factor receptor (EGFR) is overexpressed in about 90% of HNSCC tumors, molecularly targeted therapy against EGFR was thought to hold promise for HNSCC treatment. Cetuximab, a monoclonal antibody against EGFR, has been approved to treat HNSCC in the US. Still the 5-year survival rate for HNSCC is only 40-50%, despite the use of cetuximab in the clinic. The modest effect of cetuximab on survival indicates that EGFR inhibition alone will not be effective in treating HNSCC. The efficacy of cetuximab in treating HNSCC may be limited by acquired resistance or the activity of alternative dominant growth factor pathways in HNSCC. Previously, our lab has shown that in addition to EGFR signaling, the fibroblast growth factor 2 (FGF2) and its receptors (FGFRs) participate as an important autocrine signaling pathway in some gefitinib-insensitive HNSCC cell lines, further indicating that inhibiting the EGFR pathway alone will not be effective in treating HNSCC. To identify genes that change in response to EGFR-specific tyrosine kinase inhibitors (TKIs) and to FGFR-specific TKIs that may mediate acquired resistance, we performed an Affymetrix GeneChip screen in which the cell lines UMSCC25, 584-A2, and Ca9-22 were treated for 4 days with 0.3μM AZ8010, a TKI selective for FGFRs, and/or 0.1μM gefitinib, an EGFR-selective TKI. We found that in all three cell lines, TGF-β2 mRNA was upregulated following blockade of the dominant receptor pathway. Both ELISA and qRT-PCR were used to validate this induction of TGF-β2 in these three cell lines. Treatment with a small molecule inhibitor of TGF-β receptor I (TGFβRI) provided an additive reduction of clonogenic growth when combined with EGFR and/or FGFR TKIs. Additionally, silencing TGF-β2 with shRNA in UMSCC25 cells lead to an additive decrease in clonogenic growth in combination with EGFR and/or FGFR TKIs. Furthermore, we observed an induction of NF-κB activity in the cell lines following treatment with EGFR and/or FGFR TKIs. This induction was mitigated by the use of a TGF-β2 neutralizing antibody, suggesting that TGF-β2 is signaling through a non-canonical pathway in our model. In addition to observing a rapid increase in TGF-β2 expression, the cell line UMSCC25 was chronically adapted to increasing concentrations of gefitinib over the course of several months. We observed a sustained increase in TGF-β2 expression in the resistant cells compared to the control cells. This data suggests that TGF-β2 induction may provide a novel mechanism of acquired resistance to TKIs, and supports the hypothesis that combination therapy will be more effective than monotherapy in treating HNSCC. Citation Format: Emily K. Kleczko, Lydia R. Heasley, Marianne E. Marshall, Katherine R. Singleton, Lynn E. Heasley. Tyrosine kinase inhibitors induce TGF-β2 expression in head and neck squamous cell carcinoma cell lines as a mechanism of acquired resistance. [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 3387. doi:10.1158/1538-7445.AM2013-3387

Despite initial and often dramatic responses of epidermal growth factor receptor (EGFR)-addicted lung tumors to the EGFR-specific tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, nearly all develop resistance and relapse. To explore novel mechanisms mediating acquired resistance, we employed non-small-cell lung cancer (NSCLC) cell lines bearing activating mutations in EGFR and rendered them resistant to EGFR-specific TKIs through chronic adaptation in tissue culture. In addition to previously observed resistance mechanisms including EGFR-T790M ‘gate-keeper' mutations and MET amplification, a subset of the seven chronically adapted NSCLC cell lines including HCC4006, HCC2279 and H1650 cells exhibited marked induction of fibroblast growth factor (FGF) 2 and FGF receptor 1 (FGFR1) mRNA and protein. Also, adaptation to EGFR-specific TKIs was accompanied by an epithelial to mesenchymal transition (EMT) as assessed by changes in CDH1, VIM, ZEB1 and ZEB2 expression and altered growth properties in Matrigel. In adapted cell lines exhibiting increased FGF2 and FGFR1 expression, measures of growth and signaling, but not EMT, were blocked by FGFR-specific TKIs, an FGF-ligand trap and FGFR1 silencing with RNAi. In parental HCC4006 cells, cell growth was strongly inhibited by gefitinib, although drug-resistant clones progress within 10 days. Combined treatment with gefitinib and AZD4547, an FGFR-specific TKI, prevented the outgrowth of drug-resistant clones. Thus, induction of FGF2 and FGFR1 following chronic adaptation to EGFR-specific TKIs provides a novel autocrine receptor tyrosine kinase-driven bypass pathway in a subset of lung cancer cell lines that are initially sensitive to EGFR-specific TKIs. The findings support FGFR-specific TKIs as potentially valuable additions to existing targeted therapeutic strategies with EGFR-specific TKIs to prevent or delay acquired resistance in EGFR-driven NSCLC.

The efficacy of many biologically targeted cancer therapies is limited by intrinsic and acquired resistance. Head and neck squamous carcinoma (HNSCC) patients show only a 4–10% response to the epidermal growth factor receptor (EGFR) inhibitors, gefitinib and erlotinib, despite EGFR over-expression in over 90% of tumors. Our lab has previously shown that gefitinib insensitive non-small cell lung cancer (NSCLC) cell lines exhibit dominant autocrine fibroblast growth factor receptor (FGFR) signaling. RNAi-based loss-of-function screens are an important tool to identify synthetic lethal interactions between the inhibition of two genes in order to efficiently identify functional survival mechanisms and potent drug combinations. We deployed a whole genome screen to identify genes whose knockdown potentiated the inhibitory effect of the FGFR inhibitor, AZ12908010, in HNSCC cell lines. We found that multiple alternate receptors provided protection from FGFR inhibition in these cell lines, including the receptor tyrosine kinases (RTKs) epidermal growth factor receptor 2 (ErbB2) and hepatocyte growth factor receptor (Met). We validated the results of the screen by showing that specific knockdown of either ErbB2 or Met in combination with FGFR inhibitor treatment led to increased inhibition of growth relative to FGFR tyrosine kinase inhibitor (TKI) treatment alone. These results were confirmed using specific small molecule inhibitors of either ErbB2 or Met. The combination therapies did not lead to greater inhibition of growth in the cell lines where ErbB2 and Met were not identified as synthetic lethal with FGFR. We extended the study to find that the combination of knockdown of either ErbB2 or Met with FGFR inhibition decreased the growth of additional cell lines, indicating that the combination therapies could be broadly applicable. These results reveal a role for alternate RTKs in maintaining pro-growth and survival signaling in HNSCC cells in the setting of FGFR inhibition. Thus, improved therapies for HNSCC patients could involve rationally designed combinations of TKIs targeting FGFR and either ErbB family members or Met.

The identification of oncogenic molecular drivers in non-small cell lung cancer (NSCLC) has allowed biologically targeted therapies to improve clinical outcomes in the treatment of NSCLC. ALK gene rearrangements are observed in a sub-set of NSCLC patients who demonstrate high response rates to treatment with the oral kinase inhibitor, crizotinib. Unfortunately, disease progression is inevitable, either through intrinsic or acquired resistance. A recently completed series of crizotinib-resistant, ALK+ NSCLC patients by our group demonstrates two broad categories of resistance: (1) continued reliance on ALK signaling (via secondary mutations in the ALK kinase domain or ALK gene fusion copy number gain) or (2) loss of reliance on ALK signaling (e.g. via reliance on alternate oncogene signaling). Here we investigate potential therapeutic strategies when ALK signaling is retained. In an effort to minimize drug resistance to crizotinib in ALK+ NSCLC, we performed a genome-wide shRNA synthetic lethal screen to identify genes whose depletion synergizes with crizotinib in ALK+ NSCLC lines. We identified several genes involved in nucleotide synthesis and DNA metabolism, including dihydrofolate reductase (DHFR) and the trifunctional enzyme encoded by GART, which are substrates for inhibition with pemetrexed. Indeed, ALK+ NSCLC cell lines show increased sensitivity to pemetrexed in vitro. Identification of this cellular process as critical for ALK+ NSCLC is also consistent with clinical data demonstrating that patients with ALK+ NSCLC exhibit an increased clinical benefit on pemetrexed compared to other molecular subgroups of NSCLC. We also identified genes involved in chaperone function in our synthetic lethal screen. As such, we investigated whether proper folding and prevention of degradation was essential for continued reliance on ALK signaling. Both non-mutated EML4-ALK wild-type and EML4-ALK containing kinase domain mutations (including a novel mutation, G1269A) are sensitive to HSP inhibition with 17AAG. By identifying critical cellular processes in ALK+ lung cancer and employing therapies that target these pathways, improved treatment strategies can be derived to treat and prevent resistance in this molecular subtype of NSCLC. 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 5594. doi:1538-7445.AM2012-5594