Overcoming Erlotinib Resistance Research Articles

Targeting focal adhesion kinase overcomes erlotinib resistance in smoke induced lung cancer by altering phosphorylation of epidermal growth factor receptor.

EGFR-based targeted therapies have shown limited success in smokers. Identification of alternate signaling mechanism(s) leading to TKI resistance in smokers is critically important. We observed increased resistance to erlotinib in H358 NSCLC (non-small cell lung carcinoma) cells chronically exposed to cigarette smoke (H358-S) compared to parental cells. SILAC-based mass-spectrometry approach was used to study altered signaling in H358-S cell line. Importantly, among the top phosphosites in H358-S cells we observed hyperphosphorylation of EGFR (Y1197) and non-receptor tyrosine kinase FAK (Y576/577). Supporting these observations, a transcriptomic-based pathway activation analysis of TCGA NSCLC datasets revealed that FAK and EGFR internalization pathways were significantly upregulated in smoking patients, compared to the never-smokers and were associated with elevated PI3K signaling and lower level of caspase cascade and E-cadherin pathways activation. We show that inhibition of FAK led to decreased cellular proliferation and invasive ability of the smoke-exposed cells, and restored their dependency on EGFR signaling. Our data suggests that activation of focal adhesion pathway significantly contributes to erlotinib resistance, and that FAK is a potential therapeutic target for management of erlotinib resistance in smoke-induced NSCLC.

Gambogenic acid inhibits fibroblast growth factor receptor signaling pathway in erlotinib-resistant non-small-cell lung cancer and suppresses patient-derived xenograft growth

Erlotinib resistance causes a high degree of lethality in non-small-cell lung cancer (NSCLC) patients. The high expression and activation of several receptor tyrosine kinases, such as JAK/STAT3, c-Met, and EGFR, play important roles in drug resistance. The development of tyrosine kinase inhibitors is urgently required in the clinic. Our previous study found that Gambogenic acid (GNA), a small molecule derived from the traditional Chinese medicine herb gamboge, induced cell death in several NSCLC cell lines through JAK/STAT3 inhibition. In this study, we investigated the mechanism of action of GNA in erlotinib-resistant NSCLC and patient-derived cells. The inhibition of GNA on FGFR signaling pathway was examined using biochemical kinase assays. NSCLC cell lines (HCC827, HCC827-Erlotinib-resistant, and H1650) and primary cells from patients with NSCLC with clinical resistance to erlotinib were treated with GNA, erlotinib, or their combination. Both kinase assays and cell- based assays showed that GNA inhibits the phosphorylation of multiple kinases in FGFR signaling pathway in NSCLC. The combination of GNA and erlotinib significantly attenuates the tumor growth of HCC827 and erlotinib-resistant HCC827 xenografts with low toxicity. Importantly, GNA significantly suppresses tumor growth in a lung patient-derived xenograft (PDX) model with FGFR fusion and low EGFR expression. Our findings provide preclinical evidence for using GNA as an FGFR signaling pathway inhibitor to overcome erlotinib resistance in NSCLC treatment or to enhance erlotinib efficacy when used as a combined administration.

Overcoming erlotinib resistance in EGFR mutation-positive lung adenocarcinomas through repression of phosphoglycerate dehydrogenase.

How to improve the efficacy and reverse the resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib, remains a major challenge in the targeted therapy of lung adenocarcinoma with EGFR-activating mutation. Phosphoglycerate dehydrogenase (PHGDH) is the key enzyme of de novo serine biosynthesis over-expressed in various types of cancer including lung cancer. Elevated PHGDH expression is correlated with a worse overall survival in clinical lung adenocarcinoma patients. Here we investigated the role of PHGDH in lung adenocarcinoma with the acquisition of resistance to erlotinib.Methods: The necessary genes required for the acquired erlotinib resistance in lung adenocarcinoma cells were screened out by RNA-Seq analysis. Then the protein and mRNA levels of PHGDH were confirmed by immunoblotting and qRT-PCR in the erlotinib resistant cells. The effects of PHGDH inhibition or overexpression on erlotinib resistance were examined using cell culture and tumor xenograft mouse models respectively. To explore mechanism, the ROS level and DNA damage marker, γH2AX, were tested by DCFH-DA staining and immunofluorescence after PHGDH inhibition.Results: We found that PHGDH level was significantly increased in the lung adenocarcinoma PC9ER4 and HCC827ER9 cells that acquired resistance to erlotinib. Perturbation of PHGDH by siPHGDH transfection or NCT-503, a small molecular PHGDH inhibitor, synergistically augmented the tumoricidal effect and restored sensitivity to erlotinib in cell lines and xenografts. Over-expression of PHGDH caused xenografts resistant to erlotinib. Furthermore, multiple DNA damage repair pathways related genes were changed by PHGDH depletion specifically in erlotinib resistant cells. ROS stress and DNA damage marker γH2AX were enhanced by siPHGDH and NCT-503, which was reversed by NAC.Conclusion: Our study indicated that PHGDH inhibition has potential therapeutic value in lung adenocarcinoma with the acquired resistance to EGFR-TKIs.

Overcoming erlotinib resistance in EGFR mutation-positive lung adenocarcinomas through repression of phosphoglycerate dehydrogenase

Overcoming erlotinib resistance in EGFR mutation-positive lung adenocarcinomas through repression of phosphoglycerate dehydrogenase

Mimicking the BIM BH3 domain overcomes resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) are widely applied to treat EGFR-mutant non-small cell lung cancer (NSCLC). BIM is a BH3 domain-containing protein encoded by BCL2L11. Some EGFR-mutant NSCLC patients showing BIM deletion polymorphism are resistant to EGFR TKIs. We retrospectively investigated BIM deletion polymorphism in NSCLC patients, its correlation with EGFR TKI (erlotinib) resistance, and the mechanism underlying the drug resistance. Among 245 EGFR-mutant NSCLC patients examined, BIM deletion polymorphism was detected in 43 (12.24%). Median progression-free and overall survival was markedly shorter in patients with BIM deletion polymorphism than with BIM wide-type. Moreover, NSCLC cells expressing EGFR-mutant harboring BIM polymorphism were more resistant to erlotinib-induced apoptosis than BIM wide-type cells. However, combined use of erlotinib and the BH3-mimetic ABT-737 up-regulated BIM expression and overcame erlotinib resistance in EGFR-mutant NSCLC cells harboring BIM deletion polymorphism. In vivo, erlotinib suppressed growth of BIM wide-type NSCLC cell xenographs by inducing apoptosis. Combined with ABT-737, erlotinib also suppressed NSCLC xenographs expressing EGFR-mutant harboring BIM deletion polymorphism. These results indicate that BIM polymorphism is closely related to a poor clinical response to EGFR TKIs in EGFR-mutant NSCLC patients, and that the BH3-mimetic ABT-737 restores BIM functionality and EGFR-TKI sensitivity.

Abstract 2039: The use of self nano-emulsifying drug delivery systems to improve bioavailability of combination therapy and overcome erlotinib resistance in non-small cell lung cancer

Abstract Background:Erlotinib, an EGFR selective reversible inhibitor, improved response of patients diagnosed with non-small cell lung cancer (NSCLC) with 70% of patients showing significant tumor regression upon treatment. Unfortunately,most patients eventually relapse due to development of resistance. Our research goal is to quantify the effects of combination treatment of erlotinib with a glycyrrhetinic acid analogs and to improve the oral bioavailability of this treatment using self nano-emulsifying drug delivery systems. Methods: NSCLC cell lines HCC827, HCC827 (4µM erlotinib resistant), HCC827 Cl4 (second site EGFR mutated), HCC827BEAS2B, and H1975 (4µMerlotinib resistant) were treated in combination with CDODA-Me, CF3CDODA-Me and Erlotinib. The cell viability assays were performed and combination index (CI) values were calculated by isobolographic analysis. Self nano-emulsifying drug delivery systems (SNEDDs) were formulated and characterized through in vitro and in vivo studies. Results: CF3CDODA and improved efficacy for erlotinib in all cell lines with IC50 values of 6.0µM, 7.8µM, 4.6µM and 4.2±1.56µM for HCC827(RESISTANT), H1975(RESISTANT), HCC827CL4 and HCC827 respectively compared to IC50values of erlotinib alone (25µM, 21µM, 23µM, and 8µM). This was superior when compared to IC50 values of CDODA-Me combination treatment (6.66µM, 7.32µM, 12.06µM, and 5.45µM respectively). Combination treatment showed strong synergism with a constant concentration of 0.5µM CF3CDODA and CI values of 0.461, 0.548, 0.389, 0.550 for HCC827 4µM, H1975µM, HCC827CL4, and HCC827 respectively and antagonism in HCC827BEAS2B (1.508) cells. This is comparable to the synergism observed at a minimum concentration of 2µM CDODA-Me with CI values of 0.337, 0.67, 0.64, and 0.657 respectively and antagonism CI of 1.175 for HCC827BEAS2B. CF3CDODA Combination therapy showed a 2.5-fold decrease in colony formation in resistant cells making them comparable to wild type. This was comparable to CDODA-Me combination treatment with a 1.46-fold decrease in colony formation. Both combination treatments showed little effect on HCC827BEAS2B. Western Blot analysis showed decrease MET expression and decreased apoptosis inhibiting markers BCL2 and Survivin. SNEDD formulations increased maximum drug plasma concentrations for Erlotinib (281ng/ml to 442ng/ml) and circulating time as well (12hrs - 21hrs). Conclusions: Based on the results of these studies, CDODA-Me and CF3CDODA have shown to overcome erlotinib resistance in non-small cell lung cancer and when combining these treatments with the use of SNEDDS, they have shown to be a promising treatment regimen. Citation Format: Ebony Nottingham, Vasanth Sekar, Arindam Mondal, Mandip Sachdeva. The use of self nano-emulsifying drug delivery systems to improve bioavailability of combination therapy and overcome erlotinib resistance in non-small cell lung cancer [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 2039. doi:10.1158/1538-7445.AM2017-2039

Abstract 4163: Blockade of the interleukin-1 signaling pathway overcomes erlotinib resistance in head and neck cancer cells

Abstract The EGFR tyrosine kinase inhibitor erlotinib has demonstrated poor clinical response rates for head and neck squamous cell carcinoma (HNSCC) to date and the majority of respondents acquire resistance to erlotinib relatively quickly. Our previous data has shown that erlotinib activated interleukin-1 (IL-1) expression and signaling in HNSCC cells in vitro. We therefore investigated if upregulation of IL-1 signaling is involved in erlotinib resistance in HNSCC cells. We compared gene expression profiles of erlotinib-resistant (ER) and the parental erlotinib-sensitive (ES) HNSCC cell lines and observed a deregulation of the IL-1 signaling pathway in ER versus ES-HNSCC cells using microarray enrichment analysis. Secretion of IL-1 alpha (IL-1α) and IL-1 beta (IL-1β) were not significantly different in ER-SQ20B and ER-CAL 27 cells compared to their respective ES-cells, however secretion of the IL-1 receptor antagonist (IL-1RA) was significantly reduced in ER-cells compared to ES-cells. Blockade of IL-1 signaling using a recombinant IL-1R antagonist (anakinra) was able to significantly suppress the growth of ER-SQ20B but not ES-SQ20B xenografts as a single agent and in combination with erlotinib. ER-SQ20B xenografts treated with anakinra with or without erlotinib were found to be less vascularized (as determined by CD31 staining) than ER-SQ20B xenografts treated with water or erlotinib. Altogether, blockade of the IL-1 pathway using anakinra overcame erlotinib resistance in HNSCC xenografts and may represent a novel strategy to overcome EGFR inhibitor resistance for the treatment of HNSCC patients. Citation Format: Andrean L. Simons, Aditya Stanam, Katherine N. Gibson-Corley. Blockade of the interleukin-1 signaling pathway overcomes erlotinib resistance in head and neck cancer cells [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 4163. doi:10.1158/1538-7445.AM2017-4163

Combination treatment with erlotinib and ampelopsin overcomes erlotinib resistance in NSCLC cells via the Nox2-ROS-Bim pathway

Abstract Erlotinib, a tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR), has been shown to have a dramatic effect in non-small cell lung cancer (NSCLC) patients with EGFR mutation. However, the presence of primary resistance or acquired resistance to EGFR-TKI is the most common reason for switching to other anti-cancer agents. Even though there are newer agents that have activity in the presence of the T790M mutation, identification of potential agents that could overcome resistance to EGFR-TKI is still needed for the treatment of NSCLC patients. In this study, we used erlotinib-resistant NSCLC cell lines to investigate the effects of combination treatment with erlotinib and ampelopsin. To determine the effect of Nox2 and Bim on the combined treatment with erlotinib and ampelopsin-induced cell death, we transfected with Nox2 or Bim specific siRNA and performed with western blot assay for evaluation of its expression. Combined treatment with erlotinib and ampelopsin at non-cytotoxic concentrations significantly induced caspase-dependent cell death in erlotinib-resistant NSCLC cells. Furthermore, cell death resulted in the accumulation of reactive oxygen species (ROS) through upregulation of nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) expression, a direct source of ROS. The expression level of Bim increased with combination treatment, but not with either treatment alone. Here in this study, we demonstrate that the combination of erlotinib and ampelopsin induces cell death via the Nox2-ROS-Bim pathway, and ampelopsin could be used as a novel anti-cancer agent combined with EGFR-TKI to overcome resistance to erlotinib in EGFR-mutant NSCLC.

Combination treatment with erlotinib and ampelopsin overcomes erlotinib resistance in NSCLC cells via the Nox2-ROS-Bim pathway

ObjectivesErlotinib, a tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR), has been shown to have a dramatic effect in non-small cell lung cancer (NSCLC) patients with EGFR mutation. However, the presence of primary resistance or acquired resistance to EGFR-TKI is the most common reason for switching to other anti-cancer agents. Even though there are newer agents that have activity in the presence of the T790M mutation, identification of potential agents that could overcome resistance to EGFR-TKI is still needed for the treatment of NSCLC patients. Materials and methodsIn this study, we used erlotinib-resistant NSCLC cell lines to investigate the effects of combination treatment with erlotinib and ampelopsin. After treatment with either single or combination, cell viability and cell death were determined with WST-1 assay, trypan blue exclusion method, colony forming assay, annexin-V staining assay and western blot assay. The content of ROS was evaluated by FACS analysis using H2DCF-staining method. To determine the effect of Nox2 and Bim on the combined treatment with erlotinib and ampelopsin-induced cell death, we transfected with Nox2 or Bim specific siRNA and performed with western blot assay for evaluation of its expression. ResultsCombined treatment with erlotinib and ampelopsin at non-cytotoxic concentrations significantly induced caspase-dependent cell death in erlotinib-resistant NSCLC cells. Furthermore, cell death resulted in the accumulation of reactive oxygen species (ROS) through upregulation of nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) expression, a direct source of ROS. The expression level of Bim increased with combination treatment, but not with either treatment alone. ConclusionHere in this study, we demonstrate that the combination of erlotinib and ampelopsin induces cell death via the Nox2-ROS-Bim pathway, and ampelopsin could be used as a novel anti-cancer agent combined with EGFR-TKI to overcome resistance to erlotinib in EGFR-mutant NSCLC.

Overcoming erlotinib resistance with STAT3 inhibition in pancreatic cancer.

304 Background: Pancreatic ductal adenocarcinoma (PDAC) remains a major therapeutic challenge. Cytotoxic chemotherapy remains the standard approach in PDAC, but results in minimal survival benefit for patients, highlighting a desperate need for novel treatment strategies. Epidermal growth factor receptor (EGFR) is overexpressed in 25-90% of PDACs and has been shown to be an adverse prognostic marker for survival, making it a rational target. To date, the combination of the EGFR tyrosine kinase inhibitor, erlotinib, with gemcitabine remains the only approved targeted therapy based on a significant, yet modest, improved overall survival when compared to gemcitabine alone in a phase III clinical trial. We have previously shown that constitutively activated signal transducer and activator of transcription 3 (STAT3) signaling is a biomarker of resistance to both gemcitabine and erlotinib. Therefore, we hypothesized that combined STAT3 and EGFR inhibition would overcome resistance to erlotinib and gemcitabine in PDAC. Methods: Human pancreatic cancer cell lines MiaPaca2 ( KRASG12C ; TP53mut ; EGFRwt) or BxPC-3 ( KRASwt ; TP53mut ; EGFRwt) were treated in a dose dependent manner with erlotinib in the presence or absence of gemcitabine. PDAC cells were additionally treated with EC50doses of a STAT3 inhibitor (AZD1480), erlotinib, and/or gemcitabine. Lysates were then collected and western blot analysis was performed to detect total and phosphorylated extracellular signal regulated kinase (ERK) or STAT3. Results: EGFR inhibition with erlotinib, with or without gemcitabine, results in decreased ERK activation, however, causes an increased activation of STAT3 signaling in both MiaPaca2 and BxPC-3 cells. Combined STAT3 and EGFR inhibition results in sustained attenuation of both phosphorylated ERK and STAT3 levels. Conclusions: Our study demonstrates that activated STAT3 signaling appears to be a mechanism of resistance to erlotinib and gemicitabine treatment in PDAC. Combining STAT3 inhibition with EGFR inhibition overcomes this resistance.

Interleukin-1 blockade overcomes erlotinib resistance in head and neck squamous cell carcinoma

Erlotinib has demonstrated poor clinical response rates for head and neck squamous cell carcinoma (HNSCC) to date and the majority of respondents acquire resistance to erlotinib relatively quickly. To elucidate novel pathways involved in erlotinib resistance, we compared the gene expression profiles of erlotinib-resistant (ER) vs. erlotinib-sensitive (ES) HNSCC cell lines. Enrichment analysis of microarray data revealed a deregulation of the IL-1 signaling pathway in ER versus ES-HNSCC cells. Gene expression of interleukin-1 alpha (IL1A) and interleukin-1 beta (IL1B) were significantly upregulated by > 2 fold in ER-SQ20B and ER-CAL 27 cells compared to their respective ES-cells. Secretion of the IL-1 receptor antagonist (IL-1RA) was significantly reduced in ER-cells compared to ES-cells. Blockade of IL-1 signaling using a recombinant IL-1R antagonist (anakinra) was able to inhibit the growth of ER-SQ20B and ER-CAL 27 but not ES-SQ20B and ES-CAL 27 xenografts as a single agent and in combination with erlotinib. ER-SQ20B xenografts treated with anakinra ± erlotinib were found to be less vascularized than ER-SQ20B xenografts treated with water or erlotinib. Mice bearing ER-SQ20B xenografts had significantly lesser circulating levels of G-CSF and IL-1β when treated with anakinra ± erlotinib compared to those treated with water or erlotinib alone. Furthermore, augmented mRNA levels of IL1A or interleukin-1 receptor accessory protein (IL1RAP) were associated with shortened survival in HNSCC patients. Altogether, blockade of the IL-1 pathway using anakinra overcame erlotinib resistance in HNSCC xenografts and may represent a novel strategy to overcome EGFR inhibitor resistance for treatment of HNSCC patients.

Abstract A30: Modeling an immunotherapy of NK mechanism on a NSCLC patient derived xenograft

Abstract The recent clinical successes of immune checkpoint inhibitors have fueled the intense interest in novel immuno-oncology (I/O) therapeutics. The lack of relevant animal models remains a major hurdle in understanding the mechanism of action and evaluating the efficacy of such therapeutics. Patient derived xenograft (PDX), considered to most closely mimic patient tumors in both histo- and molecular pathology1,2, is however rarely used in I/O studies because it grow only in immune-compromised hosts. In reality, many PDXs grow well in nude mice where certain immune functions remain intact, excluding T-cells/T-cell functions. Therefore, PDX could still potentially be of practical use for studying T-cell independent I/O therapy. This study set out to evaluate a biologics for the treatment of a patient derived xenograft disease, by activating mouse natural killer (NK). NK and CD8 T cells are two major immune effector cell types that mediate cytotoxicity to tumor cells in vivo. One of the immunomodulatory agents, an anti-PD-L1 antibody-based IL-15 immunocytokine, was recently tested as a novel I/O treatment of cancer3. This molecule was engineered to be cross-species for both human and mouse PD-L1 and IL-15 that antagonize PD1/PD-L1 checkpoint-mediated immune suppression and also target the PD-L1-expressing tumor with IL-15 stimulated NK and CD8 T effector cells into local tumor sites, thus synergizing tumor-located anti-tumor immunity. In fact, our previous studies have demonstrated a greatly enhanced anti-tumor activity in the PDL-1-expressing syngeneic mouse tumor models via the mobilization of tumor infiltrating lymphocyte (TILs), over the PD-L1 antibody alone3. Since IL-15 also stimulates NKs in addition to T-cells, we reasoned that this bifunctional agent could also have potential activity against a PD-L1-expressing PDX in nude mice where NK remains functional. LU1901 is previously described NSCLC-PDX1, and was confirmed to express high level of PD-L1 by both RNAseq and flow analysis, after screening of a large panel of PDXs. We implanted LU1901 in Balb/c nude mice, and started to treat the mice by the bifunctional agent when the tumor reached to 150 mm3. Our result clearly demonstrated a significant inhibition of LU1901 growth by the bifunctional agent in nude mice, in the apparent absence of T-cells. When the treated tumors were examined at the termination, significantly infiltrate NK cells were found inside the treated tumors, as measured by both flow cytometry and immunohistochemistry (IHC). The number of infiltrating NK also statistically correlates to the amplitude of the tumor responses. Together, our data suggest that one of important mechanisms of action of this bifunctional agent relies on the tumor-targeting NK activation, and also that PDX could be a useful model suitable for in vivo efficacy analysis of T-cell independent immunotherapy.

Autophagy inhibition facilitates erlotinib cytotoxicity in lung cancer cells through modulation of endoplasmic reticulum stress.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have revolutionized the treatment for non-small cell lung cancer patients, but acquired resistance limit the efficiency of this treatment. As a homeostatic cellular recycling mechanism, autophagy has been proposed to participate in the EGFR-TKI resistance. However, the role of autophagy in lung cancer treatment and the underlying mechanisms have not been clarified. In this study, we found the sensitivity to erlotinib, a well-used EGFR-TKI, was correlated with basal autophagy level. Erlotinib was able to induce autophagy not only in TKI-sensitive, but also TKI-resistant cancer cells. Inhibition of autophagy significantly enhanced cytotoxicity of erlotinib in TKI-resistant cancer cells via modulation of endoplasmic reticulum (ER) stress induced apoptosis. In this process, CCAAT/enhancer binding protein homologous protein (CHOP) acted as an executioner. Downregulation of CHOP with siRNA blocked the autophagy inhibition and erlotinib co-treatment induced apoptosis and prevented cancer cells from this co-treatment-induced cell death. Our findings suggest that autophagy inhibition overcomes erlotinib resistance through modulation of ER stress mediated apoptosis.

Ezrin Enhances EGFR Signaling and Modulates Erlotinib Sensitivity in Non–Small Cell Lung Cancer Cells

Ezrin is a scaffolding protein that is involved in oncogenesis by linking cytoskeletal and membrane proteins. Ezrin interacts with epidermal growth factor receptor (EGFR) in the cell membrane, but little is known about the effects of this interaction on EGFR signaling pathway. In this study, we established the biological and functional significance of ezrin-EGFR interaction in non–small cell lung cancer (NSCLC) cells. Endogenous ezrin and EGRF interaction was confirmed by co-immunoprecipitation and immunofluorescent staining. When expression of ezrin was inhibited, EGFR activity and phosphorylation levels of downstream signaling pathway proteins ERK and STAT3 were decreased. Cell fractionation experiments revealed that nuclear EGFR was significantly diminished in ezrin-knockdown cells. Consequently, mRNA levels of EGFR target genes AURKA, COX-2, cyclin D1, and iNOS were decreased in ezrin-depleted cells. A small molecule inhibitor of ezrin, NSC305787, reduced EGF-induced phosphorylation of EGFR and downstream target proteins, EGFR nuclear translocation, and mRNA levels of nuclear EGFR target genes similar to ezrin suppression. NSC305787 showed synergism with erlotinib in wild-type EGFR-expressing NSCLC cells, whereas no synergy was observed in EGFR-null cells. Phosphorylation of ezrin on Y146 was found as an enhancer of ezrin-EGFR interaction and required for increased proliferation, colony formation, and drug resistance to erlotinib. These findings suggest that ezrin-EGFR interaction augments oncogenic functions of EGFR and that targeting ezrin may provide a potential novel approach to overcome erlotinib resistance in NSCLC cells.

Abstract C82: Induction of resistances to crizotinib in lung patient derived xenograft

Abstract Background. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase, and its mutation, over-expression and gene fusion have been associated with various cancers. EML4-ALK fusion has been confirmed to be an oncogenic driver for a small subset of the lung cancers (∼4% of NSCLC)1, and becomes an excellent drug target that led to the development of the first effective therapy, crizotinib, a tyrosine kinase inhibitor for anaplastic lymphoma kinase, for this disease 2,3. However, like any other cancer therapy so far, the treatment always led to the development of resistance, rendering them ineffective in the end4. Understanding the mechanisms causing these resistances can potentially facilitate overcoming the resistance and extend patients' life5,6. However, lack of experimental model hinders this understanding. Method. We have recently established a large collection of patient derived xenografts (∼350 PDXs)7, and we screened some of them for alk gene fusions. We then tested the positive model for sensitivity to crizothinib, and keep it under several rounds of crizotinib treatments to select resistant model. The induced resistant model was subjected to genomic analysis to identify the changes that might potentially be responsible for the resistance. Results. We identified one model, NSCLC LU1656, containing EML4-ALK fusion with elevated ALK expression. It has also been shown to respond well to crizotinib in vivo. The continued treatment eventually led to the development of resistance to crizotinib (LU2445), a situation that might occur in patients in the clinic under the same treatment. We have performed transcriptome sequencing of the parental sensitive tumor (LU1656) and the selected resistant tumor (LU2445). So far, we found both models expressed similar high levels of expression of ALK, but no additional mutations in ALK gene. There are other alterations either genetically or epigenetically, some seemingly related to the ALK signaling pathways. However, their role in resistance still need to be confirmed. Conclusions. Induced crizotinib resistance in ALK-fusion lung PDX can be useful to investigate crizotinib resistant mechanism and future drugs overcoming the resistance.

Inhibition of mitochondrial glutaminase activity reverses acquired erlotinib resistance in non-small cell lung cancer.

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib has been approved based on the clinical benefit in non-small cell lung cancer (NSCLC) patients over the past decade. Unfortunately, cancer cells become resistant to this agent via various mechanisms, and this limits the improvement in patient outcomes. Thus, it is urgent to develop novel agents to overcome erlotinib resistance. Here, we propose a novel strategy to overcome acquired erlotinib resistance in NSCLC by inhibiting glutaminase activity. Compound 968, an inhibitor of the glutaminase C (GAC), when combined with erlotinib potently inhibited the cell proliferation of erlotinib-resistant NSCLC cells HCC827ER and NCI-H1975. The combination of compound 968 and erlotinib not only decreased GAC and EGFR protein expression but also inhibited GAC activity in HCC827ER cells. The growth of erlotinib-resistant cells was glutamine-dependent as proved by GAC gene knocked down and rescue experiment. More importantly, compound 968 combined with erlotinib down-regulated the glutamine and glycolysis metabolism in erlotinib-resistant cells. Taken together, our study provides a valuable approach to overcome acquired erlotinib resistance by blocking glutamine metabolism and suggests that combination of EGFR-TKI and GAC inhibitor maybe a potential treatment strategy for acquired erlotinib-resistant NSCLC.

SAHA, an HDAC inhibitor, overcomes erlotinib resistance in human pancreatic cancer cells by modulating E-cadherin.

Pancreatic cancer is one of the most lethal cancers and remains a major unsolved health problem. Less than 20% of patients are surgical candidates, and the median survival for non-resected patients is approximately 3 to 4months. Despite the existence of many conventional cancer therapies, few targeted therapies have been developed for pancreatic cancer. Combination therapy using erlotinib and gemcitabine is an approved standard chemotherapy for advanced pancreatic cancer, but it has marginal therapeutic benefit. To try to improve the therapeutic outlook, we studied the efficacy of another combination treatment and the relevance to E-cadherin in human pancreatic cancer cells. We treated two human pancreatic cancer cell lines with the histone deacetylase inhibitor (HDACi) SAHA. Interestingly, in these Panc-1 and Capan1 cells, we observed that the expression levels of E-cadherin and phosphorylated EGFR were gradually upregulated after treatment with SAHA. Furthermore, these cells underwent induced cell death after exposure to the combination treatment of SAHA and erlotinib. In Panc-1 cells, overexpression of E-cadherin activated the phosphorylation of EGFR and increased the cell sensitivity to erlotinib. In Capan1 cells, knocking down E-cadherin decreased the expression of phosphorylated EGFR, and these cells did not respond to erlotinib. Therefore, we demonstrated the efficacy of the combined treatment with SAHA and erlotinib in human pancreatic cancer cells, and we determined that the increased efficacy was due, at least in part, to the effects of SAHA on the expression of E-cadherin. Our studies suggest that E-cadherin may be a potent biomarker for pancreatic cancer.

Abstract 1926: Patient-derived xenografts seem to have closer global expression profile to that of the patient tumors of the corresponding cancer types, than the equivalent cell lines do

Abstract Patient derived xenografts (PDXs) without in vitro manipulation are believed to mirror original patients’ histopathologic and genetic profiles, thus to be predictive surrogate models for patients, with superiority over conventional cell lines. We have built the largest and comprehensive PDX library of >1,100 models with genetic profiles of major cancer types, including some major cancer types: 200 NSLCL1, 200 CRC2, 200 gastric3, 100 HCC4, 100 pancreatic, 30 ovarian, 10 brain tumors, and many other cancer types. We set out to compare these 6 types of our PDXs with the corresponding TCGA5 patient tumor samples and CCLE6 cancer cell lines on their genomic expression by calculating pairwise Spearman rank correlation coefficient ρ, in order to further explore/confirm the similarity and difference among the three collections. PDX were profiled by both RNAseq and microarray (Affymetrix Human Genome U219 Array); CCLE were from microarray (Affymetrix Human Genome U133 Plus 2.0 Array); and TCGA were from RNAseq. For convenience, these 4 gene expression datasets are called “PDX, PDXchip, CCLE and TCGA”. Only genes common to all 4 datasets were used to compute ρ. The preliminary data seems to lead to two observations. First, for all 6 types, there is always at least one different tumor CCLE-TCGA pair that has higher ρ than the same tumor CCLE-TCGA pair. For example, the TCGA colorectal, lung, and ovarian cancers have higher ρ with CCLE pancreatic cancer than the TCGA pancreatic cancer does. In contrast, for both PDX and PDXchip, only lung and pancreatic cancers show such behavior, and notable, both to TCGA colorectal cancer. Seocnd, for same tumor type comparisons, ρ is 0.73-0.83 (average 0.772) between PDX and TCGA, 0.67-0.76 (average 0.698) between PDXchip and TCGA, 0.67-0.7 (average 0.682) between CCLE and TCGA. Third, about 20% of CCLE lung cancers have much lower ρ with TCGA, PDX, and PDXchip lung cancers than the other CCLE lung cancers. In summary, these observations show that our PDX models are close in genomic expression profile to TCGA patient tumors per tumor types specifically, and more so than CCLE cancer cell lines, which is also less specific.

Exogenous Restoration of TUSC2 Expression Induces Responsiveness to Erlotinib in Wildtype Epidermal Growth Factor Receptor (EGFR) Lung Cancer Cells through Context Specific Pathways Resulting in Enhanced Therapeutic Efficacy.

Expression of the tumor suppressor gene TUSC2 is reduced or absent in most lung cancers and is associated with worse overall survival. In this study, we restored TUSC2 gene expression in several wild type EGFR non-small cell lung cancer (NSCLC) cell lines resistant to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and analyzed their sensitivity to erlotinib in vitro and in vivo. A significant inhibition of cell growth and colony formation was observed with TUSC2 transient and stable expression. TUSC2-erlotinib cooperativity in vitro could be reproduced in vivo in subcutaneous tumor growth and lung metastasis formation lung cancer xenograft mouse models. Combination treatment with intravenous TUSC2 nanovesicles and erlotinib synergistically inhibited tumor growth and metastasis, and increased apoptotic activity. High-throughput qRT-PCR array analysis enabling multi-parallel expression profile analysis of eighty six receptor and non-receptor tyrosine kinase genes revealed a significant decrease of FGFR2 expression level, suggesting a potential role of FGFR2 in TUSC2-enhanced sensitivity to erlotinib. Western blots showed inhibition of FGFR2 by TUSC2 transient transfection, and marked increase of PARP, an apoptotic marker, cleavage level after TUSC2-erlotinb combined treatment. Suppression of FGFR2 by AZD4547 or gene knockdown enhanced sensitivity to erlotinib in some but not all tested cell lines. TUSC2 inhibits mTOR activation and the latter cell lines were responsive to the mTOR inhibitor rapamycin combined with erlotinib. These results suggest that TUSC2 restoration in wild type EGFR NSCLC may overcome erlotinib resistance, and identify FGFR2 and mTOR as critical regulators of this activity in varying cellular contexts. The therapeutic activity of TUSC2 could extend the use of erlotinib to lung cancer patients with wildtype EGFR.

Quinacrine Overcomes Resistance to Erlotinib by Inhibiting FACT, NF-κB, and Cell-Cycle Progression in Non–Small Cell Lung Cancer

Erlotinib is a tyrosine kinase inhibitor approved for the treatment of patients with advanced non-small cell lung cancer (NSCLC). In these patients, erlotinib prolongs survival but its benefit remains modest because many tumors express wild-type (wt) EGFR or develop a second-site EGFR mutation. To test drug combinations that could improve the efficacy of erlotinib, we combined erlotinib with quinacrine, which inhibits the FACT (facilitates chromatin transcription) complex that is required for NF-κB transcriptional activity. In A549 (wtEGFR), H1975 (EGFR-L858R/T790M), and H1993 (MET amplification) NSCLC cells, this drug combination was highly synergistic, as quantified by Chou-Talalay combination indices, and slowed xenograft tumor growth. At a sub-IC50 but more clinically attainable concentration of erlotinib, quinacrine, alone or in combination with erlotinib, significantly inhibited colony formation and induced cell-cycle arrest and apoptosis. Quinacrine decreased the level of active FACT subunit SSRP1 and suppressed NF-κB-dependent luciferase activity. Knockdown of SSRP1 decreased cell growth and sensitized cells to erlotinib. Moreover, transcriptomic profiling showed that quinacrine or combination treatment significantly affected cell-cycle-related genes that contain binding sites for transcription factors that regulate SSRP1 target genes. As potential biomarkers of drug combination efficacy, we identified genes that were more strongly suppressed by the combination than by single treatment, and whose increased expression predicted poorer survival in patients with lung adenocarcinoma. This preclinical study shows that quinacrine overcomes erlotinib resistance by inhibiting FACT and cell-cycle progression, and supports a clinical trial testing erlotinib alone versus this combination in advanced NSCLC.