Dual Inhibition Of Epidermal Growth Factor Receptor Research Articles

Dual Inhibition of EGFR and IGF-1R Signaling Leads to Enhanced Antitumor Efficacy against Esophageal Squamous Cancer.

Both the epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF-1R) have been implicated in the development of cancers, and the increased expression of both receptors has been observed in esophageal cancer. However, the tyrosine kinase inhibitors of both receptors have thus far failed to provide clinical benefits for esophageal cancer patients. Studies have confirmed the complicated crosstalks that exist between the EGFR and IGF-1R pathways. The EGFR and IGF-1R signals act as mutual compensation pathways, thereby conveying resistance to EGFR or IGF-1R inhibitors when used alone. This study evaluated the antitumor efficacy of the EGFR/HER2 inhibitors, gefitinib and lapatinib, in combination with the IGF-1R inhibitor, linsitinib, on the esophageal squamous cell carcinoma (ESCC). Gefitinib or lapatinib, in combination with linsitinib, synergistically inhibited the proliferation, migration, and invasion of ESCC cells, caused significant cell cycle arrest, and induced marked cell apoptosis. Their combination demonstrated stronger inhibition on the activation of EGFR, HER2, and IGF-1R as well as the downstream signaling molecules. In vivo, the addition of linsitinib to gefitinib or lapatinib also potentiated the inhibition effects on the growth of xenografts. Our results suggest the next clinical exploration of the combination of gefitinib or lapatinib with linsitinib in the treatment of ESCC patients.

Abstract 1857: Glioblastoma growth is suppressed dual inhibition of EGFR and CDK6 kinases

Abstract Glioblastoma (GBM) is a malignant brain tumor that has proven difficult to treat, despite expressing promising targets such as EGFRvIII. EGFRvIII, a mutant version of the epidermal growth factor receptor (EGFR), is constitutively active and not present in normal brain cells. The tumor specificity of EGFRvIII and the frequent EGFR amplification seen in GBM make EGFR a potentially attractive therapeutic target; however, clinical studies have shown little to no efficacy for EGFR tyrosine kinase inhibitors (TKI). One reason for this lack of efficacy may be adaptive resistance. We used RNA sequencing and multiplexed inhibitor beads with mass spectrometry (MIB-MS) to study the transcriptomes and kinomes of genetically engineered mouse astrocytes to investigate this resistance and identify potential targets for dual inhibition. Out of 329 kinases detected by MIB-MS, 76 were differentially expressed between cells with Cdkn2a deletion (“C”) and cells that also overexpressed EGFRvIII (“CEv3”). Thirty-four of these kinases were overexpressed in the CEv3 cells relative to the parental C cells (log2 fold change of 5.6, p<1x105). One of these kinases, Cdk6, is also significantly overexpressed in CEv3 cells versus cells that have a further loss of function mutation of Pten (“CEv3P”) (log2 fold change of 5.6, p<1x105). Despite this significant differential expression at the protein level, RNA expression of Cdk6 was similar between cell lines. When these cells were treated with the CDK6 inhibitor abemaciclib, CEv3 cells were found to be significantly more sensitive to inhibition than C and CEv3P cells (IC50 of 0.10 μM vs. 0.18 μM and 0.23 μM, respectively). Similarly, when cells were treated with abemaciclib in combination with the EGFR inhibitor neratinib, there was significantly higher synergy in CEv3 cells than C or CEv3P cells. Genotypically-matched patient-derived xenograft (PDX) cells were assayed for EGFR-CDK6 inhibitor synergy and showed a similar pattern of greater synergy in cells with EGFRvIII overexpression and functional PTEN than cells with EGFRvIII overexpression and PTEN loss. CEv3 and CEv3P cells were orthotopically implanted into mice and treated with neratinib, abemaciclib, or a combination. In CEv3-injected mice, combination treatment led to significantly longer survival than either single agent or control treatment. However, in CEv3P-injected mice, no survival difference was seen between any of the treatment arms. Taken together, these data provide strong evidence that CDK6 is a promising target for combination treatment with EGFR inhibitors in glioblastoma. Citation Format: Erin Smithberger, Abigail K. Shelton, Ryan E. Bash, Madison K. Butler, Alex R. Flores, Allie Stamper, Steven P. Angus, Michael P. East, Gary L. Johnson, Michael E. Berens, Frank B. Furnari, Ryan Miller. Glioblastoma growth is suppressed dual inhibition of EGFR and CDK6 kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1857.

Anti-angiogenesis revisited: reshaping the treatment landscape of advanced non-small cell lung cancer.

Although anti-angiogenic agents have been of limited use in the treatment of non-small cell lung cancer (NSCLC) until recently, further roles for the use of angiogenesis inhibition have emerged in the era of targeted therapy and immune checkpoint blockade. Given the shared common downstream signals of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) with their complementary roles in tumorigenesis and tumor angiogenesis, the dual inhibition of EGFR and VEGF pathways represents a rational strategy to maximize clinical efficacy and overcome resistance in the treatment of EGFR-mutant NSCLC. VEGF-driven angiogenesis is a potent driver of immunosuppressive tumor microenvironment (TME), with the recruited immunosuppressive cells driving angiogenesis, highlighting the interplay between the tumor vasculature and the anticancer immunity. Anti-angiogenic therapy can normalize the tumor vasculature and reprogram the TME from immunosuppressive into immunosupportive. Intensive research is under way to utilize the anti-angiogenic combination therapy to its full potential in diverse clinical settings in urgent unmet needs for the treatment of NSCLC. In this review, we present an overview of tumor angiogenesis and summarize the scientific background and preclinical and clinical evidence of anti-angiogenic therapy in combination with target therapy and immunotherapy for the treatment of NSCLC.

A bispecific decoy receptor VEGFR-EGFR/Fc binding EGF-like ligands and VEGF shows potent antitumor efficacy

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) represent two clinically validated targets for a variety of human cancers, and dual inhibition of EGFR and VEGF(R) has demonstrated superior activity to single EGFR inhibitors. This study was to construct a novel bispecific decoy receptor VEGFR-EGFR/Fc that contains Fc portion of human IgG1 acted as molecular scaffold, and the immunoglobulin-like domain 1–3 of VEGFR1 and extracellular domain of EGFR fused to the N-terminal and C-terminal of Fc, respectively, aiming at capturing the EGF-like ligands and VEGF. ELISA showed that VEGFR-EGFR/Fc bound to EGF, TGF-α and VEGF with high affinity. It displayed potent proliferation inhibitory effects on human non-small-cell lung cancer A549 cells and human umbilical vein endothelial cells revealed by MTT assays. VEGFR-EGFR/Fc significantly inhibited cell invasion and migration demonstrated by wound healing assay and transwell assay. In vivo, VEGFR-EGFR/Fc showed remarkable growth inhibition on A549 xenografts. Cell apoptosis and inhibition of angiogenesis were also observed in xenograft tumour tissues. Mechanistically, VEGFR-EGFR/Fc pre-treatment blocked the phosphorylation of EGFR and VEGFR2 and resulted in a decrease in the downstream signalling molecules, AKT, p44/42MAPK and p38MAPK. These data suggest VEGFR-EGFR/Fc would be a promising candidate for cancer targeted therapy.

Dual Targeting EGFR and STAT3 With Erlotinib and Alantolactone Co-Loaded PLGA Nanoparticles for Pancreatic Cancer Treatment.

Pancreatic cancer (PC) is one of the most common malignancies and also a leading cause of cancer-related mortality worldwide. Many studies have shown that epidermal growth factor receptor (EGFR) is highly expressed in PC, which provides a potential target for PC treatment. However, EGFR inhibitors use alone was proven ineffective in clinical trials, due to the persistence of cellular feedback mechanisms which foster therapeutic resistance to single targeting of EGFR. Specifically, the signal transducer and activator of transcription 3 (STAT3) is over-activated when receiving an EGFR inhibitor and is believed to be highly involved in the failure and resistance of EGFR inhibitor treatment. Therein, we hypothesized that dual inhibition of EGFR and STAT3 strategy could address the STAT3 induced resistance during EGFR inhibitor treatment. To this end, we tried to develop poly (lactic-co-glycolic acid) (PLGA) nanoparticles to co-load Alantolactone (ALA, a novel STAT3 inhibitor) and Erlotinib (ERL, an EGFR inhibitor) for pancreatic cancer to test our guess. The loading ratio of ALA and ERL was firstly optimized in vitro to achieve a combined cancer-killing effect. Then, the ALA- and ERL-co-loaded nanoparticles (AE@NPs) were successfully prepared and characterized, and the related anticancer effects and cellular uptake of AE@NPs were studied. We also further detailly explored the underlying mechanisms. The results suggested that AE@NPs with uniform particle size and high drug load could induce significant pancreatic cancer cell apoptosis and display an ideal anticancer effect. Mechanism studies showed that AE@NPs inhibited the phosphorylation of both EGFR and STAT3, indicating the dual suppression of these two signaling pathways. Additionally, AE@NPs could also activate the ROS-p38 axis, which is not observed in the single drug treatments. Collectively, the AE@NPs prepared in this study possess great potential for pancreatic cancer treatment by dual suppressing of EGFR and STAT3 pathways and activating ROS-responsive p38 MAPK pathway.

Dual inhibition of EGFR and IL-6-STAT3 signalling by miR-146b: a potential targeted therapy for epithelial ovarian cancer

Epidermal growth factor receptor (EGFR) signalling and the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) are aberrantly activated in ovarian cancer. However, inhibition of EGFR signalling in ovarian cancer patients resulted in a disappointing clinical benefit. In this study, we found that EGFR could activate IL-6-STAT3 pathway in ovarian cancer cells. However, we also demonstrated that EGFR knockdown could increase STAT3 phosphorylation in HO8910 and OVCAR-3 ovarian cancer cells. Interestingly, we further demonstrated that the non-coding RNA miR-146b could simultaneously block both the EGFR and IL-6-STAT3 pathways. Finally, our data demonstrated that miR-146b overexpression resulted in a greater suppression of cell migration than STAT3 pathway inhibition alone.These results suggest a complex and heterogeneous role of EGFR in ovarian cancer. Combined blockade of EGFR and IL-6-STAT3 pathways by miR-146b might be a strategy for improving the clinical benefit of targeting the EGFR pathway in ovarian cancer patients in the future.

Abstract 1675: Enhanced antitumor effect of dual EGFR and VEGFR2 inhibition in EGFR-mutated non-small cell lung cancer (NSCLC) patient-derived tumor xenograft (PDX) models

Abstract Background: NSCLC accounts for nearly 85% of all lung cancers. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are effective for EGFR mutated NSCLCs, but these drugs often stop working after EGFR acquires additional mutations. Preclinical and clinical data suggest a stronger antitumor effect with dual blockade of the EGFR and vascular endothelial growth factor (VEGF) pathways in EGFR-mutated NSCLC. RELAY, a randomized, double-blind, phase 3 trial, demonstrated superior progression-free survival (PFS) with ramucirumab (RAM) + erlotinib compared with placebo + erlotinib in patients with untreated EGFR-mutated advanced NSCLC. Additionally, RAM was tested in a Phase 1 study with the third-generation irreversible EGFR TKI osimertinib (OSI); AstraZeneca) in NSCLC patients with advanced T790M-positive EGFR mutant tumors after progression on first-line EGFR TKI therapy. OSI can be used with L858R, exon 19 del and T790M EGFR mutant tumors. We investigated the combined use of OSI+RAM in EGFR mutated NSCLC PDX mouse models. Materials and Methods: The antimurine VEGFR2 antibody DC101 (40 mg/kg, BIW) and the EGFR TKI OSI (10 mg/kg, QD) were used in this study. Twenty-seven PDX NSCLC mouse models carrying EGFR activating and/or T790M resistance mutations, commonly detected in patients with advanced NSCLC, were evaluated at 3 independent sites. Mice were divided into 4 treatment arms: vehicle (PBS), DC101, OSI and combination (DC101+OSI). Each study arm included two transplanted mice per model. Sensitivity to DC101 and OSI monotherapies, enhancement of anti-tumor activity with the combination therapy and durability of the response were evaluated. Desirability score, a weighted composite score of six features of tumor growth curves including best median % response, number of days in best response category, time to doubling, re-growth rate (last 5 observations) relative to vehicle growth rate, last observed tumor volume and last observed day, was calculated for each treatment arm. Analysis of variance was used to compare summary scores per animal across treatments. Results: OSI alone and OSI+DC101 combination therapy had stronger antitumor effects than DC101 monotherapy in significantly more models 11 vs 1 and 12 vs 0, respectively. A small but consistent shift in favor of OSI+DC101 vs OSI was observed across most of the models. Paired t-test analyses showed a moderate but significant benefit with combination OSI+DC101 vs OSI. Overall desirability score was statistically improved with OSI+DC101 compared to either single agent. Conclusions: Dual inhibition of EGFR and VEGFR2 with OSI+DC101 had a stronger antitumor effect than those of the respective monotherapies in EGFR mutated NSCLCs in vivo. Future studies will investigate the molecular mechanisms underlying the enhanced antitumor effect of OSI+DC101 combination therapy. Citation Format: Sudhakar Chintharlapalli, Anthony Fischl, Chun Ping Yu, Philip Iversen. Enhanced antitumor effect of dual EGFR and VEGFR2 inhibition in EGFR-mutated non-small cell lung cancer (NSCLC) patient-derived tumor xenograft (PDX) models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1675.

Dual inhibition of EGFR and MET by Echinatin retards cell growth and induces apoptosis of lung cancer cells sensitive or resistant to gefitinib.

Patients with non-small-cell lung cancer (NSCLC) containing epidermal growth factor receptor (EGFR) amplification or sensitive mutations initially respond to tyrosine kinase inhibitor gefitinib; however, the treatment is less effective over time. Gefitinib resistance mechanisms include MET gene amplification. A therapeutic strategy targeting MET as well as EGFR can overcome resistance to gefitinib. In the present study we identified Echinatin (Ecn), a characteristic chalcone in licorice, which inhibited both EGFR and MET and strongly altered NSCLC cell growth. The antitumor efficacy of Ecn against gefitinib-sensitive or -resistant NSCLC cells with EGFR mutations and MET amplification was confirmed by suppressing cell proliferation and anchorage-independent colony growth. During the targeting of EGFR and MET, Ecn significantly blocked the kinase activity, which was validated with competitive ATP binding. Inhibition of EGFR and MET by Ecn decreases the phosphorylation of downstream target proteins ERBB3, AKT and ERK compared with total protein expression or control. Ecn induced the G2/M cell cycle arrest, and apoptosis via the intrinsic pathway of caspase-dependent activation. Ecn induced ROS production and GRP78, CHOP, DR5 and DR4 expression as well as depolarized the mitochondria membrane potential. Therefore, our results suggest that Ecn is a promising therapeutic agent in NSCLC therapy.

MiR-30a-5p Overexpression May Overcome EGFR-Inhibitor Resistance through Regulating PI3K/AKT Signaling Pathway in Non-small Cell Lung Cancer Cell Lines.

Lung cancer is one of the most common deadly diseases worldwide, most of which is non-small cell lung cancer (NSCLC). The epidermal growth factor receptor (EGFR) mutant NSCLCs frequently respond to the EGFR tyrosine kinase inhibitors (EGFR-TKIs) treatment, such as Gefitinib and Erlotinib, but the development of acquired resistance limits the utility. Multiple resistance mechanisms have been explored, e.g., the activation of alternative tyrosine kinase receptors (TKRs) sharing similar downstream pathways to EGFR. MicroRNAs (miRNAs) are short, endogenous and non-coding RNA molecules, regulating the target gene expression. In this study, we explored the potential of miR-30a-5p in targeting the EGFR and insulin-like growth factor receptor-1 (IGF-1R) signaling pathways to overcome the drug resistance. IGF-1R is one of the tyrosine kinase receptors that share the same EGFR downstream molecules, including phosphatidylinositol 3 kinase (PI3K) and protein kinase B (AKT). In this work, an in vitro study was designed using EGFR inhibitor (Gefitinib), IGF-1R inhibitor (NVP-AEW541), and miRNA mimics in two Gefitinib-resistant NSCLC cell lines, H460 and H1975. We found that the combination of EGFR and IGF-1R inhibitors significantly decreased the phosphorylated AKT (p-AKT) expression levels compared to the control group in these two cell lines. Knockdown of phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2) had the same effect with the dual inhibition of EGFR and IGF-1R to reduce the expression of p-AKT in the signaling pathway. Overexpression of miR-30a-5p significantly reduced the expression of the PI3K regulatory subunit (PIK3R2) to further induce cell apoptosis, and inhibit cell invasion and migration properties. Hence, miR-30a-5p may play vital roles in overcoming the acquired resistance to EGFR-TKIs, and provide useful information for establishing novel cancer treatment.

Anti-EGFR monoclonal antibodies and EGFR tyrosine kinase inhibitors as combination therapy for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is characterized by overexpression of epidermal growth factor receptor (EGFR) and activation of its downstream signaling pathways. Dual targeting of EGFR using one monoclonal antibody (mAb; cetuximab or panitumumab) and one tyrosine kinase inhibitor (EGFR-TKI; gefitinib or erlotinib) is a potential therapeutic approach. We investigated the effect of these therapies in EGFR-expressing TNBC cell lines that do or do not harbor the main activating mutations of EGFR pathways. Cell lines were sensitive to EGFR-TKIs, whereas mAbs were active only in MDA-MB-468 (EGFR amplification) and SUM-1315 (KRAS and PTEN wild-type) cells. MDA-MB-231 (KRAS mutated) and HCC-1937 (PTEN deletion) cells were resistant to mAbs. The combined treatment resulted in a synergistic effect on cell proliferation and superior inhibition of the RAS/MAPK signaling pathway in mAb-sensitive cells. The anti-proliferative effect was associated with G1 cell cycle arrest followed by apoptosis. Sensitivity to therapies was characterized by induction of positive regulators and inactivation of negative regulators of cell cycle. These results suggest that dual EGFR inhibition might result in an enhanced antitumor effect in a subgroup of TNBC. The status of EGFR, KRAS and PTEN could be used as a molecular marker for predicting the response to this therapeutic strategy.

Dual inhibiting EGFR and VEGF pathways versus EGFR-TKIs alone in the treatment of advanced non-small-cell lung cancer: a meta-analysis of randomized controlled trials.

The strategy of dual inhibiting epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) pathways has been extensively investigated in advanced non-small-cell lung cancer (NSCLC), but the benefit-to-risk ratio of dual-targeted regimen versus EGFR-tyrosine kinase inhibitors (TKIs) alone is still unclear. We thus perform this meta-analysis to assess the efficacy and safety of this regimen versus EGFR-TKIs alone in those patients. Databases from PubMed, Web of Science and the Cochrane Library up to March 31, 2015 were searched to identify relevant studies. Eligible studies included prospective randomized controlled trials (RCTs) evaluating dual inhibiting EGFR and VEGF pathways versus EGFR-TKIs alone in advanced NSCLC. The endpoints were overall survival (OS), progression-free survival (PFS), objective response rate (ORR) and grade 3 or 4 adverse events. Statistical analyses were conducted by using either random effects or fixed effect models according to the heterogeneity of included studies. A total of 1918 patients with advanced NSCLC from 4 RCTs were identified for the analysis. The pooled results demonstrated that dual inhibiting EGFR and VEGF pathways significantly improved the PFS (HR 0.71, 95% CI 0.58-0.86, p<0.001) and ORR (OR 1.54, 95% CI 1.14-2.08, p=0.005) in unselected NSCLC when compared to EGFR-TKIs alone, but it did not translate into OS benefit (HR 0.94, 95% CI 0.84-1.05, p=0.24). No evidence of publication bias was observed. Our study suggests that dual inhibition of EGFR and VEGF pathways significantly improves PFS and ORR, but it does not translate into survival benefit in unselected NSCLC patients. Prospective clinical trials investigating the role of this regimen in EGFR mutation-positive NSCLC are still warranted.

Protein tyrosine phosphatase SHP-1 sensitizes EGFR/HER-2 positive breast cancer cells to trastuzumab through modulating phosphorylation of EGFR and HER-2.

BackgroundTrastuzumab resistance in HER-2 positive breast cancer cells is closely related to overexpression of both epidermal growth factor receptor (EGFR) and human epidermal receptor (HER-2). SHP-1 has been demonstrated to downregulate tyrosine kinase activity including EGFR via its phosphatase function, but its effect on HER-2 activity is still unknown. Here, we examined the hypothesis that SHP-1 enhances the anticancer efficacy of trastuzumab in EGFR/HER-2 positive breast cancer cells through combining dual inhibition of EGFR and HER-2.MethodsTrastuzumab-resistant breast cancer SKBr-3 cells were generated by long-term in vitro culture of SKBr-3cells in the presence of trastuzumab. The SHP-1 was ectopically expressed by stable transfection. The activity and expression of EGFR, HER-2, and downstream signaling pathways were tested by Western blot. Cell viability was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was examined by flow cytometry. The binding between SHP-1 and EGFR/HER-2 was evaluated by immunoprecipitation assay and bimolecular fluorescence complementation. The effects of SHP-1 on tumorigenicity and trastuzumab sensitivity were confirmed via in vivo xenograft model.ResultsTrastuzumab-resistant SKBr-3 cells showed aberrant co-expression of EGFR and HER-2. Introduction of wild-type SHP-1 inhibited cell proliferation, clone formation, and promoted the apoptosis induced by trastuzumab. Meanwhile, SHP-1 overexpression reduced phosphorylation levels of EGFR and HER-2 both in parental and trastuzumab-resistant SKBr-3 cells. In vivo study showed an increased antitumor effect of trastuzumab in SHP-1 overexpressed xenografts. At last, we discovered that SHP-1 can make complexes with both EGFR and HER-2, and both phospho-EGFR and phosphor-HER-2 levels in wild-type SHP-1 immunoprecipitates were less than those in phosphatase-inactive SHP-1 (C453S) immunoprecipitates, indicating that EGFR and HER-2 are potential substrates of SHP-1.ConclusionTaken together, we have demonstrated that the SHP-1 is a negative regulatory factor of the tyrosine kinase activity of HER-2 and EGFR through inhibiting phosphorylation. Dual targeting of EGFR and HER-2, by combining trastuzumab with SHP-1 overexpression, may improve response in HER-2 overexpressing breast cancer cells that also express high levels of EGFR.

Abstract 727: Signaling redundancy between EGFR and c-Met: molecular analysis of concurrent inhibition of c-Src and therapeutic potential against prostate cancer

Abstract Overexpression of growth factor receptors is often associated with advanced stage disease and poor prognosis. Importantly, they can interact with other growth factor receptors and non-receptor tyrosine kinases to promote proliferation, survival, invasion and metastasis. One such signaling crosstalk occurs between the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor receptor (c-Met) that are overexpressed in tumours. The amplification of c-Met in tumours overexpressing EGFR leads to signaling redundancy that causes resistance to EGFR inhibitors. Furthermore, the non-receptor tyrosine kinase c-Src has been shown to synergize with EGFR to promote tumour progression and to mediate the crosstalk between EGFR and c-Met. In order to investigate the mechanisms associated with the EGFR-c-Met-c-Src axis, we sought to identify tumour cell lines responding to the dual-inhibition of EGFR and c-Met and the triple-inhibition of EGFR, c-Met and c-Src using pharmacological inhibitions. We identified two androgen independent prostate cancer cell lines with exquisite sensitivity to EGFR-, c-Met-, c-Src-based combinations: DU145 and PC3. The results showed that the combination of crizotinib (c-Met inhibitor) + gefitinib (EGFR inhibitor) was 2-5-fold more potent (DU145 IC50 = 1.9 μM, PC3 IC50 = 1.9 μM) than the drugs alone in growth inhibition assay. Furthermore, the addition of dasatinib (c-Src inhibitor) to the crizotinib + gefitinib combination further enhanced its potency (DU145 IC50 = 0.003 μM, PC3 IC50 = 0.009 μM). When compared with individual drugs, equieffective combinations of gefitinib with crizotinib showed 2-3-fold superior potency in blocking invasion and inducing cytotoxicity. More importantly, the addition of dasatinib to the crizotinib + gefitinib combination significantly enhanced anti-invasive potency and cell-killing by apoptosis. Analysis of signaling crosstalk between the three kinases showed that dasatinib alone or in combination induced re-phosphorylation of c-Src, EGFR, c-Met and STAT3, particularly after 2-6h of drug exposure. The results indicate that these apparent compensatory re-phosphorylation mechanisms induced by dasatinib did not affect the overall potency of the triple combination. Given the role of these three tyrosine kinases in driving proliferation, invasion and survival of cancer cells, our work suggests that triple-inhibition is required for optimal antitumour activity in cells co-expressing EGFR and c-Met. Citation Format: Suman Rao, Anne-Laure Larroque-Lombard, Ben Allal, Bertrand J. Jean-Claude. Signaling redundancy between EGFR and c-Met: molecular analysis of concurrent inhibition of c-Src and therapeutic potential against prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 727. doi:10.1158/1538-7445.AM2015-727

VEGF and dual-EGFR inhibition in colorectal cancer

Epidermal growth factor receptor (EGFR), which plays an important role in tumorigenesis, is overexpressed in a high percentage of patients with late-stage colorectal cancer.1 EGFR can maintain cancer cell survival independent of its kinase activity,2 and preclinical studies have demonstrated that combining anti-EGFR antibodies and kinase inhibitors was synergistic in colon cancer cell lines.3 Angiogenesis, which plays an important role in tumor development and metastasis, is partly mediated by vascular endothelial growth factor (VEGF) and its receptor VEGFR.4 Resistance to EGFR inhibitors may be mediated, at least partly, by activating VEGF-dependent signaling, and strategies that combine anti-EGFR and anti-VEGF agents appear promising in preclinical and clinical studies.5 Preclinical studies demonstrate that EGFR signals through both kinase-dependent and independent pathways and that combining a small-molecule EGFR inhibitor, EGFR antibody, and/or anti-angiogenic agent may be synergistic.2,5 Supporting these preclinical observations, our recent report in Oncoscience demonstrates antitumor activity for the combination of cetuximab, erlotinib, and bevacizumab in heavily pretreated patients with metastatic colorectal cancer entered on a phase I trial of this triple-drug regimen.6 This is the first description of dual EGFR inhibition (erlotinib plus cetuximab) together with an anti-angiogenic agent (bevacizumab) in patients with advanced colorectal cancer. Cetuximab is a monoclonal antibody against EGFR that is approved by the Food and Drug Administration (FDA) for KRAS wild-type metastatic colorectal cancer. Erlotinib, an EGFR tyrosine kinase, is FDA-approved for non-small cell lung cancer and pancreatic adenocarcinoma. Bevacizumab, a monoclonal antibody to VEGF, is FDA-approved for metastatic colorectal cancer. The key findings of the report include description of the preliminary anti-tumor activity and safety profile of this 3-drug combination in patients with metastatic colorectal cancer. Forty-one patients were enrolled, and the study population had advanced disease, with a median of 5 prior systemic therapies. Almost all of the patients (n = 38, 93%) had previously received bevacizumab, and 33 patients (80%) had received prior cetuximab. In spite of the extensive prior treatment of the enrolled patients, stable disease (SD) of at least 6 months (n = 11) or partial response (PR) (n = 3) was achieved in 14 patients (34%), and was observed even in patients who had received prior bevacizumab and/or cetuximab or who had been treated at doses below the recommended phase 2 dose. Among the 31 patients who had received prior sequential cetuximab and bevacizumab, 10 (32%) achieved SD ≥ 6 months/PR. Of the 4 patients who had received prior concurrent bevacizumab and cetuximab, 2 achieved SD ≥ 6 months/PR. Recent investigations suggest that combining EGFR kinase inhibitors and anti-EGFR antibodies could be more effective than either alone, possibly because EGFR has the ability to maintain cancer cell survival independent of its kinase activity.1,2 A prior preclinical study combining erlotinib and cetuximab demonstrated synergistic antitumor activity in colorectal cancer, and a phase 2 trial of this combination achieved an overall response rate of 31%,3 which is similar to the rate of SD ≥ 6 months/PR observed in the present study. Prior clinical trials that combined cetuximab and bevacizumab with cytotoxic chemotherapy in patients with colorectal cancer had disappointing results, including shorter overall survival, shorter time to progression, and lower quality of life.7 It is conceivable that the chemotherapy included in such regimens may be the variable contributing to the negative outcomes in such trials, in contrast to regimens that combine anti-EGFR and anti-VEGF agents without cytotoxic chemotherapy, which may be promising and may deserve further investigation. Analysis of the side effects observed reveals that this 3-drug combination was well-tolerated in patients with metastatic colorectal cancer. The recommended phase 2 dose (RP2D) was determined to be the full FDA-approved doses of cetuximab, erlotinib, and bevacizumab. Among the 34 patients treated at the RP2D, 31 patients (91%) tolerated the treatment without drug-related dose-limiting effects. The most common treatment-related adverse events of grade 2 or higher occurring in at least 15% of patients included rash (68%) and hypomagnesemia (44%). Only two patients (5%) withdrew from the study because of toxicity, including one patient with grade 2 skin rash and another individual with grade 2 diarrhea and fatigue. Although previous studies have demonstrated correlation between rash and response to EGFR inhibitors, no such correlation or trend was observed in the current study. Overall, the findings of this study have demonstrated that dual inhibition of EGFR with erlotinib and cetuximab, combined with the VEGF antibody bevacizumab, was well-tolerated and was associated with significant antitumor activity in heavily pretreated patients with metastatic colorectal cancer. These results are promising and merit further investigation in future, larger studies.

Dual EGFR inhibition in combination with anti-VEGF treatment in colorectal cancer.

Preclinical studies demonstrate that epidermal growth factor receptor (EGFR) signals through both kinase-dependent and independent pathways and that combining a small-molecule EGFR inhibitor, EGFR antibody, and/or anti-angiogenic agent is synergistic. We conducted a dose-escalation, phase I study combining erlotinib, cetuximab, and bevacizumab. The subset of patients with metastatic colorectal cancer was analyzed for safety and antitumor activity. Forty-one patients with heavily pretreated metastatic colorectal cancer received treatment on a range of dose levels. The most common treatment-related grade ≥2 adverse events were rash (68%), hypomagnesemia (37%), and fatigue (15%). Thirty of 34 patients (88%) treated at the full FDA-approved doses of all three drugs tolerated treatment without drug-related dose-limiting effects. Eleven patients (27%) achieved stable disease (SD) ≥6 months and three (7%) achieved a partial response (PR) (total SD>6 months/PR= 14 (34%)). Of the 14 patients with SD≥6 months/PR, eight (57%) had received prior sequential bevacizumab and cetuximab, two (5%) had received bevacizumab and cetuximab concurrently, and four (29%) had received prior bevacizumab but not cetuximab or erlotinib (though three had received prior panitumumab). The combination of bevacizumab, cetuximab, and erlotinib was well tolerated and demonstrated antitumor activity in heavily pretreated patients with metastatic colorectal cancer.

Dual inhibition of EGFR and c-Met kinase activation by MJ-56 reduces metastasis of HT29 human colorectal cancer cells

Quinazolinone derivatives are known to possess anticancer activities on cell metastasis and cell death in different human cancer cell lines. Here, we studied the anti-metastasis activity and the underlying mechanisms of the novel quinazoline derivative MJ-56 (6-pyrrolidinyl-2-(3-bromostyryl)quinazolin-4-one). MJ-56 inhibited cell migration and invasion of HT29 human colorectal cancer cells by wound-healing and Matrigel-coated transwell assays in a concentration-dependent manner. MJ-56-treated cells resulted in the reduced expression of matrix metalloproteinase (MMP)-2, -7, -9 and -10 and the reduced enzymatic activities of MMP-2 and MMP-9. In contrast, MJ-56-treated cells enhanced the expression of the tissue inhibitors of metalloproteinases (TIMPs) TIMP-1 and TIMP-2. Further analyses showed that MJ-56 attenuated the activities of epidermal growth factor receptor (EGFR), c-Met and the downstream ERK-mediated MAPK and PI3K/AKT/mTOR signaling pathways, which led to decreased protein synthesis by dephosphorylating the translation initiation factors eIF-4B, eIF-4E, eIF-4G and S6 ribosomal protein. In addition, MJ-56 interfered with the NF-κB signaling via impairing PI3K/AKT activation and subsequently reduced the NF-κB-mediated transcription of MMPs. Taken together, the reduced expression of phosphor-EGFR and c-MET is chiefly responsible for all events of blocking metastasis. Our results suggest a potential role of MJ-56 on therapy of colorectal cancer metastasis.

Abstract LB-170: Preliminary report of a phase I/II trial to assess safety and tolerability of an oral Aurora kinase A inhibitor, MLN 8237 (alisertib), in combination with erlotinib in patients with recurrent or metastatic non-small cell lung cancer (NSCLC).

Abstract Introduction: NSCLC is the leading cause of cancer related death in the US. Inhibitors of the epidermal growth factor receptor (EGFR) are active in only a subset of patients. Based on our recent observation of synergistic activity of dual inhibition of EGFR and Aurora-A pathways, we are investigating the combined activity of Aurora-A inhibition (MLN8237, Alisertib, A) and an EGFR antagonist (erlotinib, E) in patients with recurrent/refractory metastatic NSCLC. Objectives: Our hypothesis is that blockade of Aurora A kinase will sensitize lung cancers to erlotinib, since the oncogenic proliferative effects of Aurora A kinase are downstream of the hyperactive EGFR-Ras-MAPK signaling axis. The primary endpoints of the Phase 1 portion include: Safety and tolerability of the combination treatment and determining the maximum tolerated dose (MTD) of A when given with E. The phase II primary endpoint is progression free survival (PFS). The secondary endpoints include PK parameters, response rate and time to treatment failure. Methods: Patients with EGFR wild type NSCLC who have progressed after at least one line of treatment were enrolled. The inclusion/exclusion criteria are as expected for a phase I/II study. This study consists of a single center Phase I run-in with the standard 3+3 design, followed by multi-center single arm phase II, with early stopping rule for futility. E is administered orally with continuous daily dosing and A is also oral, given BID for 7 days followed by 14 day rest. Treatment schema consists of 4 dose levels: 1) E 100 mg, A 30 mg, 2) E 150 mg, A 30 mg 3) E 150 mg, A 40 mg 4) E 150 mg, A 50 mg. Dosing on the Phase II portion is based on the MTD from the phase I, and will follow a two stage design that allows for early termination if the primary end point of PFS is not met. Preliminary Data: To date we have completed the first three dose levels of the phase I portion of the study. One patient is enrolled in the final level of 50 mg dose of A. MTD has not been reached. In total, 10 patients (5 male/5 female) with a median age of 62 (Range: 30 to 74) have been enrolled. All patients have had multiple prior therapies (both chemotherapy and radiation). Two patients remained on study for six cycles of therapy; four patients progressed after three cycles and four patients after two cycles (treated at lower dose levels). Three patients remain on study. Two patients have experienced dose reductions of A due to fatigue which was not defined as DLT. Other toxicities include: alopecia (one patient, 50 mg of A), transient neutropenia and anemia. At least three patients have gone to receive other forms of therapy after progression on this trial. Seven patients treated on this protocol are alive. Conclusion: The combination of E and A appears to be tolerable in patients with EGFR wild type advanced NSCLC. Dose escalation in the last cohort (50 mg po bid for seven days) continues. Citation Format: Hossein Borghaei, Ranee Mehra, Samuel Litwin, Igor Astsaturov. Preliminary report of a phase I/II trial to assess safety and tolerability of an oral Aurora kinase A inhibitor, MLN 8237 (alisertib), in combination with erlotinib in patients with recurrent or metastatic non-small cell lung cancer (NSCLC). [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-170. doi:10.1158/1538-7445.AM2013-LB-170

Activation of Multiple ERBB Family Receptors Mediates Glioblastoma Cancer Stem-like Cell Resistance to EGFR-Targeted Inhibition

Epidermal growth factor receptor (EGFR) signaling is strongly implicated in glioblastoma (GBM) tumorigenesis. However, molecular agents targeting EGFR have demonstrated minimal efficacy in clinical trials, suggesting the existence of GBM resistance mechanisms. GBM cells with stem-like properties (CSCs) are highly efficient at tumor initiation and exhibit therapeutic resistance. In this study, GBMCSC lines showed sphere-forming and tumor initiation capacity after EGF withdrawal from cell culture media, compared with normal neural stem cells that rapidly perished after EGF withdrawal. Compensatory activation of related ERBB family receptors (ERBB2 and ERBB3) was observed in GBM CSCs deprived of EGFR signal (EGF deprivation or cetuximab inhibition), suggesting an intrinsic GBM resistance mechanism for EGFR-targeted therapy. Dual inhibition of EGFR and ERBB2 with lapatinib significantly reduced GBM proliferation in colony formation assays compared to cetuximab-mediated EGFR-specific inhibition. Phosphorylation of downstream ERBB signaling components (AKT, ERK1/2) and GBM CSC proliferation were inhibited by lapatinib. Collectively, these findings show that GBM therapeutic resistance to EGFR inhibitors may be explained by compensatory activation of EGFR-related family members (ERBB2, ERBB3) enabling GBM CSC proliferation, and therefore simultaneous blockade of multiple ERBB family members may be required for more efficacious GBM therapy.

EGFR and IGF1R Inhibition in Head and Neck Cutaneous Squamous Cell Carcinoma

Objective: 1) Determine if upregulation of insulin-like growth factor receptor (IGF1R) correlates with resistance to epidermal growth factor receptor (EGFR) inhibitors in patients with head and neck cutaneous squamous cell carcinoma (cSCCHN). 2) Evaluate the effect of combined inhibition of EGFR and IGF1R with tyrosine kinase inhibitors in cSCCHN. Method: cSCC cell lines MET1, MET4, SCC12, and SCC13 were treated with the EGFR inhibitor erlotinib and/or the IGF1R inhibitor picropodophyllin, then evaluated for cell growth and expression of EGFR, IGF1R, and downstream signaling molecules. cSCCHN patient specimens were evaluated with antibodies for EGFR, IGF1R, phospho-Akt, and phospho-MAPK. Results: Comparison of normal skin to cSCCHN specimens showed overexpression of EGFR ( P = .0008) and IGF1R ( P = .041). Treatment with erlotinib or picropodophyllin inhibited receptor phosphorylation ( P &lt; .01) and showed dose-dependent inhibition of cell growth in all cell lines. At intermediate concentrations, treatment with both inhibitors inhibited cell growth more effectively than either inhibitor alone ( P &lt; .05). Dual inhibition also decreased downstream activation of both Akt ( P &lt; .006) and p42/44 MAPK ( P &lt; .01). Specimens from a patient whose cSCCHN developed resistance to an EGFR inhibitor showed upregulation of IGF1R ( P &lt; .0001) and phosphorylated Akt ( P &lt; .01) compared to this same tumor prior to treatment with EGFR inhibitor. Conclusion: Upregulation of IGF1R may be a pathway for resistance to EGFR inhibition in cSCCHN, possibly via increased Akt activation. Dual inhibition of EGFR and IGF1R inhibits cSCCHN cell growth more effectively than either inhibitor alone, suggesting that combination therapy with both inhibitors may be a promising therapeutic strategy for cSCCHN.

Dual epidermal growth factor receptor (EGFR)/insulin-like growth factor-1 receptor (IGF-1R) inhibitor: A novel approach for overcoming resistance in anticancer treatment

Small molecule inhibitors of epidermal growth factor receptors (EGFR) have been found to show a good initial response in cancer patients but during the course of treatment, patients develop resistance after a few weeks of time. Development of secondary mutations or over-activation of insulin like growth factor (IGF-1R) pathway are a few of the several mechanisms proposed to explain the resistance. To study the effect of dual inhibition of EGFR and IGF-1R in overcoming the resistance, three strategies were envisaged and are reported in this manuscript: 1) a virtual predictive tumor model, 2) in vitro experimental data using a combination of EGFR and IGF-1R inhibitors and 3) in vitro experimental data using in house dual inhibitors. Findings reported in this manuscript suggest that simultaneous inhibition of IGF-1R and EGFR either by combination of two inhibitors or by dual kinase inhibitors is more efficacious compared to single agents. In vitro cell based experiments conducted using epidermoid cancer cell line, A431 and an EGFR mutant cell line, H1975 along with virtual predictions reported here suggests that dual inhibition of EGFR and IGF-1R is a viable approach to overcome EGFR resistance.