Combined EGFR Research Articles

MET-Driven Resistance to Dual EGFR and BRAF Blockade May Be Overcome by Switching from EGFR to MET Inhibition in BRAF-Mutated Colorectal Cancer.

A patient with metastatic BRAF-mutated colorectal cancer initially responded to combined EGFR and BRAF inhibition with panitumumab plus vemurafenib. Pre-existing cells with increased MET gene copy number in the archival tumor tissue likely underwent clonal expansion during treatment, leading to the emergence of MET amplification in the rebiopsy taken at progression. In BRAF-mutated colorectal cancer cells, ectopic expression of MET conferred resistance to panitumumab and vemurafenib, which was overcome by combining BRAF and MET inhibition. Based on tumor genotyping and functional in vitro data, the patient was treated with the dual ALK-MET inhibitor crizotinib plus vemurafenib, thus switching to dual MET and BRAF blockade, with rapid and marked effectiveness of such strategy. Although acquired resistance is a major limitation to the clinical efficacy of anticancer agents, the identification of molecular targets emerging during the first treatment may afford the opportunity to design the next line of targeted therapies, maximizing patient benefit. MET amplification is here identified-clinically and preclinically-as a new mechanism of resistance to EGFR and BRAF dual/triple block combinations in BRAF-mutated colorectal cancer. Switching from EGFR to MET inhibition, while maintaining BRAF inhibition, resulted in clinical benefit after the occurrence of MET-driven acquired resistance. Cancer Discov; 6(9); 963-71. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 932.

The combined EGFR protein expression analysis refines the prognostic value of the MGMT promoter methylation status in glioblastoma

Background/aimsTo investigate the combined prognostic value of the EGFR expression level and the MGMT promoter methylation status in Glioblastoma (GB). MethodsWe assessed the EGFR protein expression level by immune-histochemical (IHC) evaluation and the MGMT promoter methylation status by Polymerase Chain Reaction (PCR) in 169 patients affected by GB. We assessed the prognostic significance of combined MGMT methylation status and EGFR expression level in terms of Overall Survival (OS) with univariate and multivariate analysis, and validated this finding using an external data set of GB patient. ResultsClustering survival analysis for the methylation status of MGMT (methMGMT/unmethMGMT) and EGFR expression (High EGFR: H-EGFR; Low EGFR: L-EGFR), identified three different prognostic groups (p=0.001), as follows. Patients with unmethMGMT/H-EGFR had the shortest survival time (median OS: 5 months) and patients co-expressing methMGMT/L-EGFR had the best prognosis (median OS: 35 months), as compared to the other two sub-groups (methMGMT/H-EGFR; unmethMGMT/L-EGFR), which had respectively median OSs of 11 and 12 months. The combined MGMT methylation and EGFR amplification status analysis showed a similar prognostic impact in an independent series, which we used for validation (p=0.001). ConclusionsThe EGFR expression evaluation refines the prognostic value of MGMT methylation status in GBs.

Abstract 4559: Sprouty2 differentially regulates signaling and phenotypic responses of glioblastoma cells to DNA damaging agents and receptor kinase inhibitors

Abstract Key to improving glioblastoma patient survival is the exploration of the signaling mechanisms that regulate cellular response to approved and investigational therapeutics, including DNA damaging agents and targeted therapeutics such as EGFR and MET kinase inhibitors. Previous work from our group demonstrated that Sprouty2 (SPRY2) surprisingly acted more as a tumor promoter than tumor suppressor in glioblastoma cell lines and tumor xenografts, with SPRY2 knockdown reducing proliferation of human glioblastoma cells and antagonizing the growth of subcutaneous tumor xenografts in mice. We hypothesized here that these effects may result from a perturbation in the cell cycle and that such effects may augment cell cycle shifts induced by DNA damaging agents or kinase inhibitors. Flow cytometry measurements indicated shifts in DNA content distribution in glioblastoma cell lines consistent with cell cycle arrest in response to SPRY2 knockdown alone, and that these shifts were substantially augmented by carboplatin or to a lesser degree by a combination of EGFR and MET inhibitors, but not by temozolomide. Western blotting indicated that SPRY2 knockdown resulted in increased phosphorylation of Ataxia Telangiectasia Mutated (ATM), a kinase involved in the DNA damage response pathway, at a serine residue (1981) that regulates the dissociation of inactivate ATM homodimers into activated monomers. In response to carboplatin, but not the other therapeutics tested, a robust increase was observed in Checkpoint kinase 1 (CHK1) phosphorylation at serine 345, which has been reported to cause cell cycle arrest. This effect was further enhanced by SPRY2 knockdown, which may help explain why the largest cell cycle perturbations were observed in SPRY2-deficient cells treated with carboplatin. Increased phosphorylation of p38 was also observed in response to carboplatin or EGFR and MET inhibitors. This may have contributed to cell cycle arrest, but may also have cooperated with the inhibition of AKT phosphorylation that occurred only in response to EGFR and MET inhibition to drive cell death, an effect that was also augmented by SPRY2 knockdown. Overall, this work has identified a small network of SPRY2-regulated signaling processes that control phenotypic responses of glioblastoma cells to different clinically relevant therapeutics and provides motivation for further study of the role of SPRY2 in glioblastoma. Citation Format: Nisha G. Sosale, Sally Shin, Matthew J. Lazzara. Sprouty2 differentially regulates signaling and phenotypic responses of glioblastoma cells to DNA damaging agents and receptor kinase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4559.

EGFR-Mediated Reactivation of MAPK Signaling Induces Acquired Resistance to GSK2118436 in BRAF V600E–Mutant NSCLC Cell Lines

Although treatment of BRAF V600E-mutant non-small cell lung cancer (NSCLC(V600E)) with GSK2118436 has shown an encouraging efficacy, most patients develop resistance. To investigate the mechanisms of acquired resistance to GSK2118436 in NSCLC(V600E), we established GSK2118436-resistant (GSR) cells by exposing MV522 NSCLC(V600E) to increasing GSK2118436 concentrations. GSR cells displayed activated EGFR-RAS-CRAF signaling with upregulated EGFR ligands and sustained activation of ERK1/2, but not MEK1/2, in the presence of GSK2118436. Treatment of GSR cells with GSK2118436 enhanced EGFR-mediated RAS activity, leading to the formation of BRAF-CRAF dimers and transactivation of CRAF. Interestingly, sustained activation of ERK1/2 was partly dependent on receptor-interacting protein kinase-2 (RIP2) activity, but not on MEK1/2 activity. Combined BRAF and EGFR inhibition blocked reactivation of ERK signaling and improved efficacy in vitro and in vivo Our findings support the evaluation of combined BRAF and EGFR inhibition in NSCLC(V600E) with acquired resistance to BRAF inhibitors. Mol Cancer Ther; 15(7); 1627-36. ©2016 AACR.

Abstract B14: Targeting the intratumoral heterogeneity of receptor tyrosine kinases in breast cancer

Abstract Breast cancers display a remarkable phenotypic diversity that is exploited to promote both tumor progression and therapeutic resistance. Recent studies in several types of cancer have highlighted the significance of intratumoral heterogeneity on both innate and acquired resistance to tyrosine kinase inhibitors (TKIs). Tumor plasticity is supported by the heterogeneous expression of receptor tyrosine kinases (RTKs) and the robustness that the overlapping signaling networks provide. Therefore a thorough understanding of the intratumoral heterogeneity is necessary for the development of effective therapeutic strategies. The receptor tyrosine kinase MET is overexpressed in 20-30% of breast cancers and correlates with poor patient outcome. Previously, we determined that high MET expression correlated with ER-/ERBB2- and basal like breast cancers. These results and the efficacy of MET inhibitors in other cancers suggest that MET may be an effective clinical target for aggressive breast cancer subtypes. Recent studies have exposed interactions between MET and the ERBB receptor family in the progression and therapeutic resistance of several cancers. Since MET, ERBB2, and EGFR are known to be highly expressed in aggressive breast cancer subtypes, it is critical that we understand the relationships between these receptors in order to develop effective treatment strategies. We are investigating the relationship between MET and ERBB receptor signaling in the progression and resistance of ERBB2+ and triple-negative breast cancer (TNBC). We observe that there is a large subset of ERBB2+ breast cancers that express MET and contain MET+/ERBB2+ subpopulations. In a MET+/ERBB2+ breast cancer cell line, MET depletion results in increased ERBB2 activation whereas, ERBB2 depletion results in increased MET activation. Therefore, ERBB2+ breast cancers with MET+ subpopulations may have an innate resistance to ERBB2 inhibition and may benefit from combined MET and ERBB2 inhibition. In TNBC, we observe heterogeneous expression of MET and EGFR. We have developed patient-derived xenografts (PDX) from primary and metastatic TNBCs that have diverse patterns of MET and EGFR expression/activation. In these TNBC PDX models we observe varied responses to monotherapy with MET inhibitors MGCD265 and crizotinib and the EGFR inhibitor erlotinib. Interestingly, therapeutic response to MET inhibition does not correlate with protein expression levels. In all studies, we observe significantly increased efficacy of combination therapy with MET and EGFR inhibition and a decrease in response variability. Examination of phospho-MET localization in treated tumorgrafts revealed that MET and EGFR inhibition induces distinct phospho-MET localization changes. We also observe that in the residual resistant cells phospho-MET is highly expressed in cells with mitotic bodies. We are currently performing phospho-proteomic analysis to determine the effect of MET and/or EGFR inhibition on RTK signaling networks including ERK and AKT pathways. These results will identify proteomic signatures that represent MET and/or EGFR activation/inhibition and sensitivity or resistance to monotherapy or combined MET and EGFR inhibition. Overall these studies give us a more comprehensive view of TNBC network signaling, the level of RTK heterogeneity within TNBC, and the efficacy of MET and/or EGFR inhibition on TNBC progression. Citation Format: Elizabeth A. Tovar, Erik S. Linklater, Curt J. Essenburg, Zach Madaj, David M. Cherba, Mary E. Winn, Hasan Korkaya, Julie L. Boerner, Carrie R. Graveel. Targeting the intratumoral heterogeneity of receptor tyrosine kinases in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B14.

Abstract 337: Mechanism of TKI resistance and role of epithelial mesenchymal transition in NSCLC

Abstract Current EGFR/c-Met tyrosine kinase inhibitor (TKI) combination therapies are shown to be susceptible to acquired resistance in the majority of NSCLC patients. To understand how cells develop EGFR/c-Met TKI resistance, we used three model NSCLC cell lines: H2170, H3255 and H1975. H2170 cell line express high levels of EGFR and c-Met and are EGFR wild-type. The H3255 cell line is positive for an EGFR TKD mutation, L858R, and is sensitive to initial TKI treatment. The H1975 cell line is positive for two EGFR TKD mutations, T790M and L858R, which confer resistance to erlotinib. Previous studies in our lab have shown that activation of alternative signaling pathways lead to the development of TKI resistance. In the wild type TKI resistant cells, H2170 erlotinib resistant (ER) and H2170 SU11274 resistant(SR), we observed activation of Wnt and mTOR pathway along with increased accumulation of β-catenin in the nucleus. However, in H1975 cells with mutated EGFR (L858R and T790M), we observed activation of only mTOR pathway. β-catenin is associated with Epithelial-Mesenchymal Transition (EMT), during which cells lose tight junction proteins such E-Cadherin and gain expression of transcriptional proteins such as β-catenin and ZEB1, which may lead to aberrant activation of Wnt signaling pathway and increased motility and invasion. Activation of mTOR pathway leads to increased expression of p70S6K and 4E-BP1 which are key translational regulators. We found increased accumulation of active β-catenin in the nucleus by 1.9-2.5 folds and 2.9-3.1 folds in H2170 ER and SR cells, respectively, compared to H2170 parental cells. In H2170 ER cells we observed modulations in key EMT-related proteins, N-Cadherin (2-3.3 fold) and Claudin-1 (2.5-14 fold) were upregulated and Snail was downregulated 1.7-2.5 fold in the presence and absence of erlotinib and EGF, when compared to H2170 parental cells. Similarly in H2170 SR cells we observed upregulation of N-Cadherin (1.3-2.3 fold) and Zeb-1 (1.2-1.4 fold) and downregulation of ZO-1 (1.4-1.9 fold) and E-Cadherin (1.5-2.0 fold) in the presence and absence of HGF and SU11274 when compared to H2170 parental cells. Morphological changes indicative of EMT were detected using immunostaining with Vimentin and E-Cadherin antibodies, which displayed increased Vimentin filaments (2-fold) and loss of E-Cadherin (5-10 fold) in H2170 ER cells when compared to H2170 parental cells. In H1975 cells we observed that proteins related to mTOR signaling pathway such as p-mTOR, p-GSK3-β, p-4EBP1 and p-p70S6K were upregulated 2.5 fold, 4-5 fold, 2.2-2.4 fold, and 4.4-10.4 fold, respectively, when compared to H3255 cells in the presence of erlotinib. Based on these results, we treated H1975 cells with combination of EGFR and mTOR inhibitors. We observed that 1 μM everolimus with 2.5 μM erlotinib synergistically inhibited cell growth by 53%. These studies may provide clinicians with novel targets for improving treatment options for future NSCLC patients. Citation Format: Ichwaku Rastogi, Gregory M. Botting, Andrew Webb, Brian L. Webb, Marie C. Nlend, Neelu Puri. Mechanism of TKI resistance and role of epithelial mesenchymal transition in NSCLC. [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 337. doi:10.1158/1538-7445.AM2015-337

Abstract LB-004: Mouse PDX Trial Suggests Combination Efficacy of Raf and EGFR Inhibition in Colorectal Cancer with BRaf or KRas mutation

Abstract MAPK activation through KRas, NRas or BRaf mutation occurs in approximately 70% of colorectal cancer patients. Due to their epithelial origin, colorectal tumors generally have high levels of EGFR expression and activation. EGFR therapies such as cetuximab are effective for treatment of a subset of colorectal cancer, particularly patients with wild type (WT) KRas. EGFR signaling is also recently identified as a key resistance mechanism in BRaf mutant colorectal cancer to BRaf inhibitors. In this study, we have genetically characterized 78 patient-derived xenograft (PDX) models of colorectal tumors, and conducted an “n = 1” (single mouse per treatment group) trial in these PDX models with cetuximab, LSN3074753, a pan-Raf and Raf dimer inhibitor, and their combination in collaboration with Oncotest GmbH and Champions Oncology. Among these 78 PDX models, 42 (53.8%) have a KRas mutation, 12 (15.4%) have BRaf V600E or an atypical BRaf mutation, and 26 (33.3%) are WT KRas and BRaf. Consistent with clinical results, cetuximab is primarily active in WT KRas and BRaf PDX models, with disease control rate (DCR) of 53.8% (14/26) in this subgroup. These results suggest that the mouse n = 1 PDX trial paradigm could reliably predict clinical results. For pan-Raf and Raf dimer inhibitor LSN3074753, it is active in a subset of PDX models, particularly those with BRaf or KRas mutation(s), with DCR of 21.2% among models with a KRas or BRaf mutation. Importantly, a synergistic effect is observed when cetuximab and LSN3074753 are combined for treatment of these 78 PDX models. The overall DCR in the combination arm is 50% (39/78), while cetuximab or LSN3074753 alone has an overall DCR of 24 or 18%, respectively. Further statistical analyses reveal that BRaf mutations including V600E or other atypical mutations (G469E, G76E, G596V, G203V, etc) are the best predictor of combination synergy, and are significantly associated with synergistic effect with a p value of 0.004. In models with BRaf mutations, the combination arm has a DCR of 50% (6/12), whereas cetuximab or LSN3074753 alone has a DCR of 8.3 or 17%, respectively. BRaf or KRas mutations are also significantly associated with combination synergy with p value of 0.01. Among 42 KRas mutation models, LSN3074753 or cetuximab alone has a DCR of 21.4 or 16.7%, and the combination arm has a DCR of 43%. Overall, these results indicate that combination of EGFR and Raf inhibition by cetuximab and a pan-Raf inhibitor has the potential for treatment of colorectal cancer patients with BRaf or KRas mutation. Citation Format: Yung-mae M. Yao, Gregory P. Donoho, Philip W. Iversen, Yue Wang Webster, Yong Gang Yue, James R. Henry, Gregory D. Plowman, Sheng-Bin Peng. Mouse PDX Trial Suggests Combination Efficacy of Raf and EGFR Inhibition in Colorectal Cancer with BRaf or KRas mutation. [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 LB-004. doi:10.1158/1538-7445.AM2015-LB-004

Abstract 2845: Co-acquisition of T790M and EMT in resistant EGFR mutant non-small cell lung cancer can be overcome by combined irreversible EGFR and BCL-XL inhibition

Abstract The development of acquired drug resistance limits the effectiveness of targeted therapies for lung cancer. In 50% of patients with EGFR mutant non-small cell lung cancer (NSCLC), acquired resistance to EGFR inhibitors can be attributed to the development of a secondary T790M mutation that prevents drug binding. Third generation irreversible EGFR inhibitors that effectively inhibit EGFR with T790M are currently under clinical development. Using patient-derived EGFR mutant NSCLC models of T790M+ acquired resistance, we observed that the presence of a mesenchymal phenotype was associated with a decreased response to subsequent irreversible EGFR inhibitor therapy. Epithelial-to-mesenchymal transformation (EMT) led to suppression of pro-apoptotic signaling and a blunted apoptotic response to EGFR inhibition. Combination BCL-XL and irreversible EGFR inhibitor therapy restored the apoptotic response and resulted in regressions of T790M+ resistant tumors that exhibited poor response to single agent irreversible EGFR inhibitors. This suggests that combining BCL-XL and irreversible EGFR inhibitors may be an effective treatment strategy for T790M+ resistant EGFR mutant lung cancers with suppressed apoptotic signaling due co-acquisition of a mesenchymal phenotype. Citation Format: Aaron N. Hata, Matthew J. Niederst, Hannah L. Archibald, Hillary Mulvey, Jungoh Ham, Maria Gomez-Caraballo, Anuj Kalsy, Anthony C. Faber, Jeffrey Engelman. Co-acquisition of T790M and EMT in resistant EGFR mutant non-small cell lung cancer can be overcome by combined irreversible EGFR and BCL-XL inhibition. [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 2845. doi:10.1158/1538-7445.AM2015-2845

Abstract 4147: Synergistic effect of combined EGFR and Src inhibitor in colon cancer cell migration

Abstract EGFR signaling plays the important role in cancer cell proliferation and cell migration. Cetuximab which is the antibody against EGFR was approved for colon cancer treatment. Recently, Src inhibitor, dasatinib has been demonstrated to improve drug sensitivity to cetuximab in KRAS mutant colorectal cancer cell growth. Src is a non-receptor tyrosine kinase which is necessary for full activation of EGFR and also functions in focal adhesion kinase activity in cell migration, therefore, the combination of cetuximab and dasatinib may has effect on colon cell migration. In this study, cetuximab and dasatinib were applied to SW480 colon cell which is KRAS mutant cell line. Cell viability and cell migration were analyzed, localization of Src and focal adhesion kinase (FAK) were observed by confocal microscopy. In the result, Combination of 5μM cetuximab and 40 nM dasatinib treatment resulted in synergistic effect on cell viability. However, when cetuximab combined with 25nM dasatinib was applied in trans-well migration assay, the synergistic inhibition on cell migration was observed. The immunofluorescence confocal microscopic image demonstrated cetuximab treatment induced phospho-Src accumulation on the cell membrane and colocalized with phospho-FAK. When the combination of 5μM cetuximab and 25 uM dasatinib were applied in the cell, the membrane accumulation of phospho-Src and Phospho-FAK were decreased. Taken together, cetuximab combined with dasatinib has synergistic efficacy in colon cancer cell proliferation, however, cetuximab combined with lower concentration of dasatinib (25nM) suppressed cell migration by inhibited FAK membrane localization through Src inactivation. Citation Format: Yi-Che Wu, Wan-Chen Wei, Wei-Ting Chao. Synergistic effect of combined EGFR and Src inhibitor in colon cancer cell migration. [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 4147. doi:10.1158/1538-7445.AM2015-4147

Improved diagnostics targeting c-MET in non-small cell lung cancer: expression, amplification and activation?

BackgroundSeveral c-MET targeting inhibitory molecules have already shown promising results in the treatment of patients with Non-small Cell Lung Cancer (NSCLC). Combination of EGFR- and c-MET-specific molecules may overcome EGFR tyrosine kinase inhibitor (TKI) resistance. The aim of this study was to allow for the identification of patients who might benefit from TKI treatments targeting MET and to narrow in on the diagnostic assessment of MET.Methods222 tumor tissues of patients with NSCLC were analyzed concerning c-MET expression and activation in terms of phosphorylation (Y1234/1235 and Y1349) using a microarray format employing immunohistochemistry (IHC). Furthermore, protein expression and MET activation was correlated with the amplification status by Fluorescence in Situ Hybridization (FISH).ResultsCorrelation was observed between phosphorylation of c-MET at Y1234/1235 and Y1349 (spearman correlation coefficient rs = 0.41; p < 0.0001). No significant correlation was shown between MET expression and phosphorylation (p > 0.05). c-MET gene amplification was detected in eight of 214 patients (3.7 %). No significant association was observed between c-MET amplification, c-MET protein expression and phosphorylation.ConclusionOur data indicate, that neither expression of c-MET nor the gene amplification status might be the best way to select patients for MET targeting therapies, since no correlation with the activation status of MET was observed. We propose to take into account analyzing the phosphorylation status of MET by IHC to select patients for MET targeting therapies. Signaling of the receptor and the activation of downstream molecules might be more crucial for the benefit of therapeutics targeting MET receptor tyrosine kinases than expression levels alone.

Acquired Resistance to the Mutant-Selective EGFR Inhibitor AZD9291 Is Associated with Increased Dependence on RAS Signaling in Preclinical Models.

Resistance to targeted EGFR inhibitors is likely to develop in EGFR-mutant lung cancers. Early identification of innate or acquired resistance mechanisms to these agents is essential to direct development of future therapies. We describe the detection of heterogeneous mechanisms of resistance within populations of EGFR-mutant cells (PC9 and/or NCI-H1975) with acquired resistance to current and newly developed EGFR tyrosine kinase inhibitors, including AZD9291. We report the detection of NRAS mutations, including a novel E63K mutation, and a gain of copy number of WT NRAS or WT KRAS in cell populations resistant to gefitinib, afatinib, WZ4002, or AZD9291. Compared with parental cells, a number of resistant cell populations were more sensitive to inhibition by the MEK inhibitor selumetinib (AZD6244; ARRY-142886) when treated in combination with the originating EGFR inhibitor. In vitro, a combination of AZD9291 with selumetinib prevented emergence of resistance in PC9 cells and delayed resistance in NCI-H1975 cells. In vivo, concomitant dosing of AZD9291 with selumetinib caused regression of AZD9291-resistant tumors in an EGFRm/T790M transgenic model. Our data support the use of a combination of AZD9291 with a MEK inhibitor to delay or prevent resistance to AZD9291 in EGFRm and/or EGFRm/T790M tumors. Furthermore, these findings suggest that NRAS modifications in tumor samples from patients who have progressed on current or EGFR inhibitors in development may support subsequent treatment with a combination of EGFR and MEK inhibition.

Estimated glomerular filtration rate and albuminuria for prediction of cardiovascular outcomes: a collaborative meta-analysis of individual participant data

BackgroundThe utility of estimated glomerular filtration rate (eGFR) and albuminuria for cardiovascular prediction is controversial.MethodsWe meta-analyzed individual-level data from 24 cohorts (with a median follow-up time longer than 4 years, varying from 4.2 to 19.0 years) in the Chronic Kidney Disease Prognosis Consortium (637,315 participants without a history of cardiovascular disease) and assessed C-statistic difference and reclassification improvement for cardiovascular mortality and fatal and non-fatal cases of coronary heart disease, stroke, and heart failure in 5-year timeframe, contrasting prediction models consisting of traditional risk factors with and without creatinine-based eGFR and/or albuminuria (either albumin-to-creatinine ratio [ACR] or semi-quantitative dipstick proteinuria).FindingsThe addition of eGFR and ACR significantly improved the discrimination of cardiovascular outcomes beyond traditional risk factors in general populations, but the improvement was greater with ACR than with eGFR and more evident for cardiovascular mortality (c-statistic difference 0.0139 [95%CI 0.0105–0.0174] and 0.0065 [0.0042–0.0088], respectively) and heart failure (0.0196 [0.0108–0.0284] and 0.0109 [0.0059–0.0159]) than for coronary disease (0.0048 [0.0029–0.0067] and 0.0036 [0.0019–0.0054]) and stroke (0.0105 [0.0058–0.0151] and 0.0036 [0.0004–0.0069]). Dipstick proteinuria demonstrated smaller improvement than ACR. The discrimination improvement with kidney measures was especially evident in individuals with diabetes or hypertension but remained significant with ACR for cardiovascular mortality and heart failure in those without either of these conditions. In participants with chronic kidney disease (CKD), the combination of eGFR and ACR for risk discrimination outperformed most single traditional predictors; the c-statistic for cardiovascular mortality declined by 0.023 [0.016–0.030] vs. <0.007 when omitting eGFR and ACR vs. any single modifiable traditional predictors, respectively.InterpretationCreatinine-based eGFR and albuminuria should be taken into account for cardiovascular prediction, especially when they are already assessed for clinical purpose and/or cardiovascular mortality and heart failure are the outcomes of interest (e.g., the European guidelines on cardiovascular prevention). ACR may have particularly broad implications for cardiovascular prediction. In CKD populations, the simultaneous assessment of eGFR and ACR will facilitate improved cardiovascular risk classification, supporting current CKD guidelines.FundingUS National Kidney Foundation and NIDDK

A phase I (Ph1) study of dasatinib (D) with cetuximab (Cet) /radiation (IMRT) +/- cisplatin (P) in stage II, III/IV head and neck squamous cell carcinoma (HNSCC).

e17036 Background: In preclinical HNSCC models, combined EGFR and Src inhibition with radiation has additive and possibly synergistic effects. We conducted a 3+3 standard Ph1 trial to determine the...

Dual inhibition of EGFR and MET induces synthetic lethality in triple-negative breast cancer cells through downregulation of ribosomal protein S6

Triple-negative breast cancer (TNBC) exhibits innate resistance to the EGFR inhibition despite high level expression of EGFR. Recently, we found that the proliferation of basal-like (BL) subtype TNBC cells is synergistically inhibited by combination of EGFR and PI3K/AKT inhibitors. On the contrary, TNBC cells of mesenchymal stem-like (MSL) subtype are resistant to these combinations. To identify potential synthetic lethal interaction of compounds for treatment of MSL subtype TNBC cells, we performed MTT screening of MDA-MB-231 cells with a small library of receptor tyrosine kinase inhibitors (RTKIs) in the presence of gefitinib, an EGFR inhibitor. We identified MET inhibitors as potent RTKIs that caused synthetic lethality in combination with gefitinib in MDA-MB-231 cells. We demonstrated that combination of a MET inhibitor SU11274 with various EGFR inhibitors resulted in synergistic suppression of cell viability (in MTT assay) and cell survival (in colony formation assay) of MSL subtype TNBC cells. We further demonstrated that SU11274 alone induced G2 arrest and gefitinib/SU11274 combination sustained the SU11274-induced G2 arrest in these cells. In addition, SU11274/gefitinib combination synergistically reduced the level of ribosomal protein S6 (RPS6) in MSL subtype TNBC cells. In addition, knockdown of RPS6 itself, in both HS578T and MDA-MB-231, markedly reduced the proliferation of these cells. Taken together, our data suggest that dual targeting of EGFR and MET inhibits the proliferation of MSL subtype TNBC cells through down-regulation of RPS6.

A paradigm shift: Cancer therapy with peptide-based B-cell epitopes and peptide immunotherapeutics targeting multiple solid tumor types: Emerging concepts and validation of combination immunotherapy

There is a recognizable and urgent need to speed the development and application of novel, more efficacious anti-cancer vaccine therapies that inhibit tumor progression and prevent acquisition of tumor resistance. We have created and established a portfolio of validated peptide epitopes against multiple receptor tyrosine kinases and we have identified the most biologically effective combinations of EGFR (HER-1), HER-2, HER-3, VEGF and IGF-1R peptide vaccines/mimics to selectively inhibit multiple receptors and signaling pathways. The strategy is based on the use of chimeric conformational B-cell epitope peptides incorporating “promiscuous” T-cell epitopes that afford the possibility of generating an enduring immune response, eliciting protein-reactive high-affinity anti-peptide antibodies as potential vaccines and peptide mimics that act as antagonists to receptor signaling that drive cancer metastasis. In this review we will summarize our ongoing studies based on the development of combinatorial immunotherapeutic strategies that act synergistically to enhance immune-mediated tumor killing aimed at addressing mechanisms of tumor resistance for several tumor types.

A Phase II Study of Sorafenib Combined With Cetuximab in EGFR-Expressing, KRAS-Mutated Metastatic Colorectal Cancer

BackgroundMutations in the KRAS gene predict for resistance to anti-epidermal growth factor receptor (EGFR) therapies, including cetuximab. Upregulation of vascular endothelial growth factor (VEGF)-A has been implicated in resistance to anti-EGFR treatment. Abrogation of the VEGF and RAS/RAF/MEK/ERK pathways has the potential to restore cetuximab sensitivity. Patients and MethodsAdult patients with histologically documented, measurable, EGFR-expressing, KRAS-mutated metastatic colorectal cancer (mCRC) that had progressed after 5-fluorouracil–based regimens were treated with sorafenib 400 mg orally twice daily and intravenous cetuximab weekly in 28-day cycles. The primary endpoint was the response rate (complete response, partial response, and stable disease at 4 cycles). The secondary endpoints included plasma biomarker analysis of angiogenic cytokines and correlative imaging studies with dynamic contrast-enhanced magnetic resonance imaging and zirconium 89-panitumumab. ResultsOf the 30 patients enrolled, 26 were evaluable for response. Of the 26 patients evaluated, 4 had stable disease at 4 cycles and 1 had stable disease at 8 cycles. The median progression-free survival was 1.84 months. The common toxicities were rash, diarrhea, and liver enzyme elevations. Of the angiogenic cytokines evaluated, only the placental growth factor increased significantly with treatment (P < .0001). No pharmacodynamic parameters were associated with the treatment response. ConclusionWe report the results of a trial that combined cetuximab and sorafenib for the treatment of KRAS-mutated mCRC, with correlative imaging studies and pharmacodynamic angiogenic cytokine profiling as downstream markers of EGFR and VEGF receptor (VEGFR) signaling. No objective responses were observed. Additional development of biomarkers for patient selection is needed to evaluate combined EGFR and VEGFR blockade as a therapeutic option in KRAS-mutated CRC.

Abstract 5471: Contextual synthetic lethality in human triple negative breast cancer cells involving a novel interaction between DNA repair proteins EGFR, PARP1, and BRCA1

Abstract Background/Objectives: Few therapeutic options are effective for triple negative breast cancers (TNBCs) necessitating novel approaches. We previously reported a novel synthetic lethality with combined EGFR and PARP inhibition due to a homologous recombination (HR) repair defect induced EGFR inhibition. The mechanism of the HR deficiency involved disruption of EGFR/BRCA1 interaction and sequestration of EGFR and BRCA1 to the cytosol. In this study, we have extended these findings to encompass multiple subtypes of triple negative breast cancer and EGFR inhibitors. Additionally, we further investigated the mechanisms by which EGFR regulates HR DNA repair. Methods: TNBC cells, including MDA-MB-231 (mesenchymal stem-like), MDA-MB-468 (basal-like), HCC1187 (immunomodulatory), BT549 (mesenchymal), MDA-MB-453 (luminal androgen receptor), were assessed for cytotoxicity and cell viability following EGFR inhibition (lapatinib, erlotinib, or cetuximab), PARP inhibition, or both by colony formation or ATPlite assays. MALDI-TOF mass spectroscopy was utilized to ascertain protein-protein interaction. To confirm interaction data and determine subcellular location we utilized immunoprecipitation and subcellular fractionation. To determine if interactions were dependent on DNA, lysates were treated with Ethidium Bromide and DNAse prior to immunoprecipitation. Results: EGFR inhibition with lapatinib, cetuximab, or erlotinib induced a contextual synthetic lethality with the PARP inhibitor, veliparib, in vitro in multiple TNBC subtypes, including MDA-MB-231 (mesenchymal stem-like), MDA-MB-468 (basal-like), HCC1187 (immunomodulatory), BT549 (mesenchymal), MDA-MB-453 (luminal androgen receptor). Interestingly, mass spectroscopy reaffirmed EGFR interaction with BRCA1 but also identified a novel interaction between EGFR and PARP1. These interactions were not found upon EGFR inhibition with lapatinib. Furthermore, we validated with immunoprecipitation and in vitro protein binding assays EGFR, BRCA1, and PARP1 protein-protein interactions that were disrupted upon EGFR inhibition. Additionally, these proteins were found to interact in the nucleus, independent of DNA. Lastly, EGFR inhibition also reduced nuclear EGFR and BRCA1 without significant changes in PARP1 localization. Conclusions: These results reveal a contextual synthetic lethality between combined EGFR and PARP inhibition in multiple subtypes of TNBC, suggesting potential generalizability of this combination in patients with TNBC. Furthermore understanding the mechanism by which EGFR regulates DNA repair may provide novel targets to confer other contextual synthetic lethalities that may be exploited to treat the aggressive TNBCs. Citation Format: Jennifer Anne Stanley, Somaira Nowsheen, Tiffiny Cooper, Andres Forero, Alfred LoBuglio, Eddy Yang. Contextual synthetic lethality in human triple negative breast cancer cells involving a novel interaction between DNA repair proteins EGFR, PARP1, and BRCA1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5471. doi:10.1158/1538-7445.AM2014-5471

Abstract 1832: Combined EGFR and MEK inhibition prevents the emergence of drug resistance in EGFR mutant non-small cell lung cancer (NSCLC)

Abstract Background: EGFR inhibitors are effective clinical therapies for EGFR mutant NSCLC, but efficacy is limited by the development of acquired drug resistance. The mechanisms of resistance include secondary mutations in EGFR (T790M) and activation of compensatory signaling pathways (MET, IGFR and AXL). Mutant selective EGFR inhibitors (WZ4002, CO-1686 and AZD9291) are effective in preclinical and clinical models of EGFR T790M. Our lab has demonstrated that reactivation of ERK signaling is a mechanism of resistance to WZ4002 (WZ) which could be reversed or prevented by using a combination of WZ and MEK inhibitors. Given the broad array of possible EGFR inhibitor resistance mechanisms, we evaluated whether WZ combined with the MEK inhibitor trametinib (TRA) can prevent the emergence of resistance in vitro and in vivo. Methods and Results: We developed a plate based resistance assay in which 350 cells/well are plated in 96 well plates and treated weekly. This system allows for evaluation of drug efficacy over weeks of treatment and can be used to measure both time to development and frequency of drug resistance. Given that ERK reactivation can cause resistance to WZ, we evaluated WZ, TRA or the combination thereof (WZ/TRA) using PC9 cells. Single agents led to 100% resistance in 2-3 weeks, versus 5% with WZ/TRA in 24 weeks. We then tested WZ/TRA in 5 additional EGFR TKI sensitive cell lines, including several known to develop acquired resistance to gefitinib (HCC827, MET amplification; HCC4006, EMT or FGFR activation; HCC2279; FGFR activation; H3255, T790M; HCC2935). In all cases, WZ/TRA significantly reduced the emergence of drug resistant clones compared to each single agent. In models with established drug resistance mechanisms, WZ/TRA was effective in 3/3 models with T790M mutations, but not (0/5) in models with established non-T790M resistance mechanisms. In models where WZ/TRA was effective, the combination led to increased apoptosis compared to single agents and effective inhibition of ERK signaling. We also evaluated the effectiveness of the WZ/TRA combination in vivo using EGFR L858R/T790M genetically engineered mouse model and PC9GR4 (T790M+) xenografts. Combination WZ/TRA treatment prevented tumor outgrowth for 24 weeks in 5 EGFR L858R/T790M GEMM mice whereas individual treatments did not. Additionally, we found that WZ/TRA combination treatment, but not single agents, could cure 7/15 xenograft tumors as assessed by lack of tumor regrowth after treatment cessation. Conclusion: Our studies suggest that the combination of a mutant selective EGFR inhibitor and a MEK inhibitor can prevent the emergence of both T790M and non-T790M mediated drug resistance mechanisms. This strategy is more effective at preventing (6/6) than treating (3/8) cancers with established drug resistance mechanisms and should be evaluated in clinical trials in EGFR TKI naïve EGFR mutant NSCLC patients. Citation Format: Erin M. Tricker, Chunxiao Xu, Dalia Ercan, Atsuko Ogino, Kwok-kin Wong, Pasi Janne. Combined EGFR and MEK inhibition prevents the emergence of drug resistance in EGFR mutant non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1832. doi:10.1158/1538-7445.AM2014-1832

Abstract 1690: Attacking EGFR mutant lung cancer by combined EGFR and c-Met inhibition

Abstract In lung adenocarcinomas, targeted therapy with the EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib is associated with longer progression free survival (PFS). However, despite the initial success, all patients progress with a median PFS of 12 to 16 months. Acquired resistance is driven by the occurrence of a secondary EGFR mutation (T790M) in about 50% of the cases and by c-Met amplification in 5 to 10 % of the cases. We report the activity of a new reversible and ATP-competitive c-Met inhibitor, EMD1214063, in the setting of primary lung adenocarcinomas harboring EGFR activating mutations and in two models of acquired resistance. EMD1214063 showed to be specifically active in c-Met amplified and c-Met dependent cells in a large panel of genotypically characterized lung cancer cell lines. In the context of acquired resistance, we studied the effect of EMD1214063 alone and in combination of afatinib, an irreversible EGFR TKI, in PC9 cells (EGFR Exon19del), H1975 cells (EGFR L858R and T790M), HCC827 (EGFR Exon19del, not c-Met amplified) and in HCC827GR cells (EGFR Exon19del and c-Met amplified). Treatment of HCC827GR cells with EMD1214063 resulted in substantial growth inhibition and induced apoptosis. The in vitro results showed a significant synergistic effect of the combination of EMD121039 and afatinib in the induction of growth inhibition in the context of c-Met amplification and EGFR T790M mutation. Combination treatment robustly suppressed expression of downstream pErk and pAkt, showing efficient suppression of PI3K and MAPK signaling. To determine whether the antitumor activity of the combination observed in vitro might also be apparent in vivo, we injected these cells in nude mice to elicit the formation of solid tumors. Mice were treated with EMD121043 (25mg/kg), afatinib (10mg/kg) or the combination of both. In H1975 xenografts only the combination therapy could significantly reduce tumor growth, thus confirming the results obtained in vitro. In the c-Met amplified setting, we treated mice harboring HCC827, HCC827GR and mixed xenografts. HCC827-driven tumors strongly responded to both afatinib monotherapy and to the combination, leading to complete remission within 40 days of therapy. HCC827/HCC827GR mixed xenografts containing 0.1%, 1% and 10% of HCC827GR cells partially responded to afatinib monotherapy; however, growth was completely abolished by combination therapy. Treatment with EMD1214063 led to significant growth reduction in mice harboring HCC827GR tumors. In this model, only the combination therapy led to massive tumor shrinkage. Assessment of cell proliferation in vivo by [18F]FLT-PET showed a decline in FLT-uptake of 40% in both the combination therapy and the EMD1214063 monotherapy. In conclusion, the combination of EMD1214063 and afatinib promote tumor regression in erlotinib acquired resistant lung cancers driven by EGFR L858R/T790M or EGFR Exon19 del/c-Met amplification. Citation Format: Sandra Ortiz-Cuarán, Jakob Schöttle, Ilona Dahmen, Martin Peifer, Caroline Wieczoreck, Mirjam Koker, Michaela A. Ihle, Alexandra Florin, Berit Pinther, Lukas C. Heukamp, Roland T. Ullrich, Roman K. Thomas. Attacking EGFR mutant lung cancer by combined EGFR and c-Met inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1690. doi:10.1158/1538-7445.AM2014-1690

Abstract 1842: Synergistic inhibition of ALK inhibitor-resistant lung cancer cells by dual-targeting EGFR and ALK pathways

Abstract Background: Despite the excellent initial responses of ALK-rearranged lung cancer to the ALK tyrosine kinase inhibitors (TKIs), most patients develop acquired resistance through several mechanisms, including ALK amplification, mutation or activation of alternative signaling pathways. In this study, we investigated the effect of dual-targeting EGFR and ALK pathways in NSCLC cells that are resistant to ALK inhibitors. Methods: The cell proliferation, apoptosis and cell cycle analysis were measured by spectrophotometry and flow cytometry. The combination effect of EGFR TKI (afatinib or dacomitinib) and ALK TKI (crizotinib or LDK378) was evaluated using a combination index (CI). We analyzed EGFR ligand levels by ELISA and expression of ALK, EGFR and its downstream molecules were detected by Western blot assay after exposure to TKIs. Results: NCI-H3122CR1 that was resistant to crizotinib and NCI-H3122 cell lines became resistant to LDK378 through the step-wise increase of the drug, resulting in H3122CR1LR1 and H3122LR1 cells, respectively. The resistant cell lines exhibited 10-14-fold higher IC50 values for crizotinib or LDK378 compared with parental cells. The genetic alterations of ALK or EGFR genes were not found in H3122CR1, H3122LR1, and H3122CR1LR1 cells. We analyzed EGFR signaling pathways in these cells to determine their roles in mediating ALK inhibitor resistance. We observed that H3122CR1, H3122LR1 and H3122CR1LR1 cells had higher phospho-EGFR than parental cells. In these cells, mRNA and protein levels of amphiregulin and EGF were up-regulated. Combined treatment with EGFR TKI and LDK378 synergistically inhibited cell growth (CI&amp;lt;1) and induced apoptosis in H3122LR1 and H3122CR1LR1 cells. Similar results were observed in H3122CR1 and SNU-2535 harboring ALK G1269A mutation cells, when exposed to afatinib and crizotinib. The combination of EGFR and ALK TKIs decreased the phosphorylation of several downstream molecules in H3122LR1 and H3122CR1LR1 cells, whereas EGFR or ALK TKI alone did not reduce the phosphorylation levels. Conclusion: Our results suggest that dual inhibition of ALK and EGFR effectively abrogate ALK and its downstream signals and induce apoptosis in ALK TKIs-resistant, ALK-rearranged NSCLC cells. Therefore, dual-targeting of EGFR and ALK might be a promising strategy for ALK-rearranged NSCLC patients who showed acquired resistance to crizotinib and/or LDK378. Citation Format: Soyeon Kim, Tae Min Kim, Yong-Oon Ahn, Bhumsuk Keam, Se-Hoon Lee, Dae Seog Heo, Dong-Wan Kim. Synergistic inhibition of ALK inhibitor-resistant lung cancer cells by dual-targeting EGFR and ALK pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1842. doi:10.1158/1538-7445.AM2014-1842