Epidermal Growth Factor Receptor Inhibitor Erlotinib Research Articles

Long-term treatment with valganciclovir improves lentiviral suicide gene therapy of glioblastoma.

Suicide gene therapy for malignant gliomas has shown encouraging results in the latest clinical trials. However, prodrug application was most often restricted to short-term treatment (14 days), especially when replication-defective vectors were used. We previously showed that a substantial fraction of herpes simplex virus thymidine kinase (HSV-TK) transduced tumor cells survive ganciclovir (GCV) treatment in an orthotopic glioblastoma (GBM) xenograft model. Here we analyzed whether these TK+ tumor cells are still sensitive to prodrug treatment and whether prolonged prodrug treatment can enhance treatment efficacy. Glioma cells positive for TK and green fluorescent protein (GFP) were sorted from xenograft tumors recurring after suicide gene therapy, and their sensitivity to GCV was tested in vitro. GBM xenografts were treated with HSV-TK/GCV, HSV-TK/valganciclovir (valGCV), or HSV-TK/valGCV + erlotinib. Tumor growth was analyzed by MRI, and survival as well as morphological and molecular changes were assessed. TK-GFP+ tumor cells from recurrent xenograft tumors retained sensitivity to GCV in vitro. Importantly, a prolonged period (3 mo) of prodrug administration with valganciclovir (valGCV) resulted in a significant survival advantage compared with short-term (3 wk) application of GCV. Recurrent tumors from the treatment groups were more invasive and less angiogenic compared with primary tumors and showed significant upregulation of epidermal growth factor receptor (EGFR) expression. However, double treatment with the EGFR inhibitor erlotinib did not increase therapeutic efficacy. Long-term treatment with valGCV should be considered as a replacement for short-term treatment with GCV in clinical trials of HSV-TK mediated suicide gene therapy.

Automatic 3D Nonlinear Registration of Mass Spectrometry Imaging and Magnetic Resonance Imaging Data.

Multimodal integration between mass spectrometry imaging (MSI) and radiology-established modalities such as magnetic resonance imaging (MRI) would allow the investigations of key questions in complex biological systems such as the central nervous system. Such integration would provide complementary multiscale data to bridge the gap between molecular and anatomical phenotypes, potentially revealing new insights into molecular mechanisms underlying anatomical pathologies presented on MRI. Automatic coregistration between 3D MSI/MRI is a computationally challenging process due to dimensional complexity, MSI data sparsity, lack of direct spatial-correspondences, and nonlinear tissue deformation. Here, we present a new computational approach based on stochastic neighbor embedding to nonlinearly align 3D MSI to MRI data, identify and reconstruct biologically relevant molecular patterns in 3D, and fuse the MSI datacube to the MRI space. We demonstrate our method using multimodal high-spectral resolution matrix-assisted laser desorption ionization (MALDI) 9.4 T MSI and 7 T in vivo MRI data, acquired from a patient-derived, xenograft mouse brain model of glioblastoma following administration of the EGFR inhibitor drug of Erlotinib. Results show the distribution of some identified molecular ions of the EGFR inhibitor erlotinib, a phosphatidylcholine lipid, and cholesterol, which were reconstructed in 3D and mapped to the MRI space. The registration quality was evaluated on two normal mouse brains using the Dice coefficient for the regions of brainstem, hippocampus, and cortex. The method is generic and can therefore be applied to hyperspectral images from different mass spectrometers and integrated with other established in vivo imaging modalities such as computed tomography (CT) and positron emission tomography (PET).

Abstract P3-10-15: Evaluating contribution of hyperactive c-Met and ErbB signaling to tumor progression in mouse breast tumor xenografts: An in vivo study of c-Met and ErbB targeted therapies

Abstract Background: Hyperactive c-Met signaling, including cross-talk between c-Met and ErbB family receptors, is suspected of contributing to tumor progression in a variety of cancer types. Clinical trials evaluating c-Met inhibitors alone and in combination with other targeted therapies, have produced mostly negative results in patients with amplified c-Met. This suggests other biological factors, such as c-Met and ErbB signaling activity, may be important to measure when identifying patients eligible for c-Met therapies. To measure the c-Met and ErbB signaling activity of a patient's live tumor cells, a new assay using an impedance biosensor, the CELx multi-pathway signaling function (CELx MP) test, was developed. The CELx MP test measures a patient's ex vivo live tumor cell response in real-time to specific ErbB and c-Met agonists to diagnose breast cancer tumors with hyperactive HER1, HER2, HER3, HER4, and c-MET signaling activity. In this study, to further elucidate the role of c-Met signaling and its potential involvement with ErbB signaling as a cancer driver, we studied in vivo response to a c-Met inhibitor (tepotinib), an EGFR inhibitor (erlotinib), a pan-HER inhibitor (neratinib), and a combination of these therapies using breast tumor xenograft models. Methods: HCC1954, a HER2+ cell line with hyperactive c-Met and EGFR signaling and normal HER3 and HER2-driven signaling, according to the CELx MP test, was studied. Sixty 4-5-week-old female NSG mice were injected with two million cells. Mice were randomly assigned to either a control group that received Captisol or one of five treatment groups that received either neratinib, tepotinib, erlotinib, erlotinib and tepotinib, or neratinib and tepotinib for 17 days. Results: The most effective treatment was the combination of neratinib plus tepotinib, where the average tumor size reduction relative to the control group was 71% (p=0.0003). The average tumor size in the neratinib plus tepotinib treated group was 37% smaller (p=0.049) than the neratinib treated group, and 67% smaller (p=0.0026) than the tepotinib treated group. In the erlotinib plus tepotinib treatment group, the average tumor size was 51% smaller than the control group tumors, but the difference was not statistically significant (p=0.11). No significant difference in tumor size was found between the control group and the erlotinib or tepotinib treated groups. Conclusions: The results demonstrate that hyperactive and coincident c-Met and EGFR signaling contributes to the progression of certain breast cancers. This breast cancer sub-type is more responsive to treatment with a c-Met inhibitor plus a pan-HER inhibitor than a c-Met inhibitor plus an EGFR inhibitor or any of the single agents studied. This suggests that when hyperactive c-Met signaling and a hyperactive ErbB family member is present in a patient's tumor, each of the ErbB pathways as well as the c-Met pathway must be inhibited to treat the tumor most effectively. These findings provide strong evidence that HER2-negative breast cancer patients with coincident hyperactive c-MET and ErbB signaling may respond to treatment with a combination of pan-HER and c-Met inhibitors. Citation Format: Laing LG, Burns DJ, Khan S, MacNeil IA, Rich BE, Kharbush SM, Soltani SM, Sullivan BF. Evaluating contribution of hyperactive c-Met and ErbB signaling to tumor progression in mouse breast tumor xenografts: An in vivo study of c-Met and ErbB targeted therapies [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-15.

Abstract P6-18-35: A phase 1 study of erlotinib and metformin in advanced triple negative breast cancer

Abstract Background: The epidermal growth factor receptor (EGFR) is frequently overexpressed in triple negative breast cancer (TNBC). However, EGFR inhibitors have not shown efficacy as monotherapy in TNBC. One strategy for overcoming resistance to EGFR inhibition is concomitant inhibition of downstream signaling. Metformin is a LKB1-dependent AMPK activator that inhibits both MAPK and AKT signaling. The combination of the EGFR inhibitor erlotinib and metformin synergistically induces apoptosis in TNBC cell lines and decreases tumor burden in PTEN-null EGFR-amplified mouse xenograft models. We evaluated the combination of erlotinib and metformin in a phase 1 study of patients with advanced TNBC. Methods: Patients with advanced TNBC who had received at least one prior line of therapy for metastatic disease were eligible. Erlotinib dose was fixed at 150mg daily. Metformin dose escalation was planned according to a 3+3 design, beginning at 850mg BID and escalating to 850mg TID. One de-escalation to 500mg BID was allowed. Dose-limiting toxicities (DLT) were assessed during the first five weeks of therapy. The primary objectives were to determine the maximum tolerated dose (MTD) of metformin with fixed dose erlotinib and to determine the potential for clinical benefit. Secondary endpoints were response rate, stable disease rate, and progression free survival. Pre- and on-treatment skin biopsies were collected to determine the effect of the study drugs on their respective cell signaling targets, particularly EGFR, AMPK, and mTOR. Results: Between March 2013 and May 2015, nine patients were screened and eight were enrolled. Median age was 48 years (range 37-79). Median number of prior therapies for metastatic disease was 2.5 (range 1-6). No DLT events were reported in either of the dose escalation cohorts during the DLT assessment period. AEs occurring in three or more patients and all grade III AEs are reported in Table 1. Grade III diarrhea despite maximum supportive care required dose reduction of metformin from 850mg TID to 850mg BID in one patient. Grade III rash led to study withdrawal in one patient. No grade IV AEs were reported. Per RECIST v1.1, the best observed response was stable disease in two patients (25%). Median time on study was 2.0 months (range 1.2-3.0). Skin biopsy marker assessment is ongoing and will be reported. Conclusion: The combination of erlotinib and metformin was generally well tolerated in a population of pre-treated metastatic TNBC patients. No unexpected toxicities occurred. While no responses were achieved, stable disease was observed in patients who received this non-chemotherapy combination. Adverse EventsEventMetformin 850mg BID n=3Metformin 850mg TID n=5All patients n=8 Number of patients (percent) All gradesGrade IIIAll gradesGrade IIIAll gradesGrade IIIRash3 (100)1 (33.3)5 (100)08 (100)1 (12.5)Diarrhea3 (100)05 (100)2 (40.0)8 (100)2 (25.0)Weight loss1 (33.3)05 (100)06 (75.0)0Dry skin1 (33.3)05 (100)06 (75.0)0Nausea2 (66.7)03 (60.0)05 (62.5)0Vomiting1 (33.3)03 (60.0)04 (50.0)0Dry mouth1 (33.3)03 (60.0)04 (50.0)0Dysgeusia1 (33.3)02 (40.0)03 (37.5)0Increased creatinine2 (66.7)01 (20.0)03 (37.5)0Fatigue1 (33.3)02 (40.0)03 (37.5)0Anorexia1 (33.3)02 (40.0)03 (37.5)0Hyponatremia1 (33.3)1 (33.3)001 (12.5)1 (12.5) Citation Format: Fenn KM, Maurer MA, Lee SM, Crew KD, Trivedi MS, Accordino MK, Hershman DL, Kalinsky K. A phase 1 study of erlotinib and metformin in advanced triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-18-35.

Insight into binding mechanisms of EGFR allosteric inhibitors using molecular dynamics simulations and free energy calculations

Lung cancer is the leading cause of cancer death, and epidermal growth factor receptor (EGFR) kinase domain mutations are a common cause of non-small-cell lung cancer (NSCLC), a major subtype of lung cancers. Patients harboring most of these mutations respond well to the EGFR inhibitors Gefitinib and Erlotinib initially, but soon develop resistance to them due to the emergence of the gatekeeper mutation T790M. The new-generation inhibitors such as AZD9291, HM61713, CO-1686 and WZ4002 can overcome T790M through covalent binding to Cys 797, but ultimately lose their efficacy upon the emergence of the C797S mutation that abolishes the covalent bonding. Allosteric inhibitors EAI001 and EAI045 are a new type of EGFR inhibitors that bind to EGFR away from the ATP-binding site and not relying on Cys 797. In this study, molecular dynamics simulations and free energy calculations were carried out on EAI001 and EAI045 in complex with EGFR, revealing the detailed inhibitory mechanism of EAI001 and EAI045 as EGFR allosteric inhibitor, which was expected to provide a basis for rational drug design of the EGFR allosteric inhibitors.Communicated by Ramaswamy H. Sarma

Monensin inhibits cell proliferation and tumor growth of chemo-resistant pancreatic cancer cells by targeting the EGFR signaling pathway

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly malignancies with <5% five-year survival rate due to late diagnosis, limited treatment options and chemoresistance. There is thus an urgent unmet clinical need to develop effective anticancer drugs to treat pancreatic cancer. Here, we study the potential of repurposing monensin as an anticancer drug for chemo-resistant pancreatic cancer. Using the two commonly-used chemo-resistant pancreatic cancer cell lines PANC-1 and MiaPaCa-2, we show that monensin suppresses cell proliferation and migration, and cell cycle progression, while solicits apoptosis in pancreatic cancer lines at a low micromole range. Moreover, monensin functions synergistically with gemcitabine or EGFR inhibitor erlotinib in suppressing cell growth and inducing cell death of pancreatic cancer cells. Mechanistically, monensin suppresses numerous cancer-associated pathways, such as E2F/DP1, STAT1/2, NFkB, AP-1, Elk-1/SRF, and represses EGFR expression in pancreatic cancer lines. Furthermore, the in vivo study shows that monensin blunts PDAC xenograft tumor growth by suppressing cell proliferation via targeting EGFR pathway. Therefore, our findings demonstrate that monensin can be repurposed as an effective anti-pancreatic cancer drug even though more investigations are needed to validate its safety and anticancer efficacy in pre-clinical and clinical models.

Integrated mapping of pharmacokinetics and pharmacodynamics in a patient-derived xenograft model of glioblastoma

Therapeutic options for the treatment of glioblastoma remain inadequate despite concerted research efforts in drug development. Therapeutic failure can result from poor permeability of the blood-brain barrier, heterogeneous drug distribution, and development of resistance. Elucidation of relationships among such parameters could enable the development of predictive models of drug response in patients and inform drug development. Complementary analyses were applied to a glioblastoma patient-derived xenograft model in order to quantitatively map distribution and resulting cellular response to the EGFR inhibitor erlotinib. Mass spectrometry images of erlotinib were registered to histology and magnetic resonance images in order to correlate drug distribution with tumor characteristics. Phosphoproteomics and immunohistochemistry were used to assess protein signaling in response to drug, and integrated with transcriptional response using mRNA sequencing. This comprehensive dataset provides simultaneous insight into pharmacokinetics and pharmacodynamics and indicates that erlotinib delivery to intracranial tumors is insufficient to inhibit EGFR tyrosine kinase signaling.

Neurotensin promotes cholangiocarcinoma metastasis via the EGFR/AKT pathway

Cholangiocarcinoma (CCA) is a fatal disease with increasing morbidity and poor prognosis due to poor response to conventional chemotherapy or radiotherapy. Neurotensin (NTS) has long been recognized as an important factor in the central nervous system and as an endocrine agent in the peripheral circulation via NTS receptor (NTSR) mediated actions. In recent years, NTS has been implicated in the carcinogenesis of numerous cancers; however, its role in cholangiocarcinoma remains obscure. Here, we observed the expression of NTS in cholangiocarcinoma vs. non-cancerous tissues and found that up-regulation of NTS facilitated cholangiocarcinoma cell metastasis and down-regulation of NTS inhibited their migration ability. Mechanistically, NTS drove cholangiocarcinoma cell metastasis via the EGFR/AKT pathway. Both the PI3-K inhibitor LY294002 or EGFR inhibitor Erlotinib stopped the discrepant metastatic capacity between NTS-depleted cholangiocarcinoma cells and control cells, further confirming that EGFR/AKT was required in NTS-promoted cholangiocarcinoma cell metastasis. More importantly, overexpression of NTS predicted poor prognosis of CCA patients. In summary, NTS could promote cholangiocarcinoma cells metastasis by amplifying EGFR/AKT signaling and may therefore be useful to predict patient prognosis.

Drug-Sensitivity Screening and Genomic Characterization of 45 HPV-Negative Head and Neck Carcinoma Cell Lines for Novel Biomarkers of Drug Efficacy.

There is an unmet need for effective targeted therapies for patients with advanced head and neck squamous cell carcinoma (HNSCC). We correlated gene expression, gene copy numbers, and point mutations in 45 human papillomavirus-negative HNSCC cell lines with the sensitivity to 220 anticancer drugs to discover predictive associations to genetic alterations. The drug response profiles revealed diverse efficacy of the tested drugs across the cell lines. Several genomic abnormalities and gene expression differences were associated with response to mTOR, MEK, and EGFR inhibitors. NOTCH1 and FAT1 were the most commonly mutated genes after TP53 and also showed some association with response to MEK and/or EGFR inhibitors. MYC amplification and FAM83H overexpression associated with sensitivity to EGFR inhibitors, and PTPRD deletion with poor sensitivity to MEK inhibitors. The connection between high FAM83H expression and responsiveness to the EGFR inhibitor erlotinib was validated by gene silencing and from the data set at the Cancer Cell Line Encyclopedia. The data provide several novel genomic alterations that associated to the efficacy of targeted drugs in HNSCC. These findings require further validation in experimental models and clinical series. Mol Cancer Ther; 17(9); 2060-71. ©2018 AACR.

Chemotherapeutic Treatments Increase PD-L1 Expression in Esophageal Squamous Cell Carcinoma through EGFR/ERK Activation

The current study reveals the clinicopathological association of PD-L1 in Hong Kong esophageal squamous cell carcinoma (ESCC) patients and the differential regulation of PD-L1 by standard first-line chemotherapy in ESCC. Immunohistochemical analysis of tissue microarray data from 84 Hong Kong ESCC patients shows that PD-L1 was expressed in 21% of the tumors. Positive PD-L1 staining was significantly associated with later disease stage (stages III and IV) (P value = .0379) and lymph node metastasis (P value = .0466) in the Hong Kong cohort. Furthermore, PD-L1 expression was significantly induced in ESCC cell lines after standard chemotherapy treatments, along with EGFR and ERK activation in both in vitro studies and the in vivo esophageal orthotopic model. The endogenous expression of PD-L1 was reduced by treatment with an EGFR inhibitor (erlotinib) or by the knockdown of EGFR. Moreover, the upregulation of PD-L1 by chemotherapy was also attenuated by the treatment with erlotinib and a MAPK/MEK inhibitor (AZD6244), suggesting that PD-L1 is regulated by the EGFR/ERK pathway in ESCC. The regulation of PD-L1 by the EGFR pathway was further supported by the correlation of PD-L1 and EGFR expression observed in the commercially available tissue microarray set (P value = .028). Taken together, the current study was the first to demonstrate the upregulation of PD-L1 by chemotherapy in ESCC and its regulation through the EGFR/ERK pathway. The results suggest the potential usefulness of combined conventional chemotherapy together with anti–PD-L1 immunotherapy to achieve better treatment outcome.

Abstract A48: MET and EGFR interaction promotes acquired resistance to kinase inhibition in TNBC

Abstract Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and the molecular heterogeneity within TNBC heightens the challenge of developing effective targeted therapies. Receptor tyrosine kinases (RTKs), in particular MET and EGFR, are promising therapeutic targets for TNBC due to their high expression in multiple molecular TNBC subtypes and the tendency for cancers to become “kinase addicted.” We and others have demonstrated that MET is highly expressed in TNBC and its expression correlates with poor prognosis. EGFR expression is also elevated in up to 72% of TNBCs and correlates with poor prognosis in TNBC patients. Recently, we demonstrated that MET and EGFR are coordinately and highly expressed across all TNBC subtypes and the efficacy of combined MET and EGFR inhibition in TNBC patient-derived xenograft (PDX) models. Even though MET and EGFR receptors are actionable targets due to their high activity in TNBC, crosstalk between MET and EGFR has been implicated in therapeutic resistance to kinase inhibitors in several cancer types and needs to be evaluated in TNBC. There is strong evidence demonstrating the critical role of redundant RTK signaling networks in resistance to tyrosine kinase inhibitors (TKIs). Specifically, crosstalk between MET and EGFR has been implicated in therapeutic resistance to EGFR or MET inhibitors in colon, gastric, and lung cancers. Since MET and EGFR are highly expressed in a substantial proportion of TNBCs, we used TNBC PDX models and TNBC cell lines to interrogate mechanisms of resistance to MET and EGFR kinase inhibition. We hypothesized that coexpression, interaction, and activation of MET and EGFR promote acquired TKI resistance and an adaptive kinome response in TNBC. Using TNBC PDX models that highly express both MET and EGFR, we evaluated MET and EGFR activity, localization, and interaction after 3 days or 3 weeks of treatment with MET (glesatinib, crizotinib) and/or EGFR inhibitors (erlotinib). After 3 weeks of dual MET and EGFR inhibition in TNBC PDXs, only a small population of “resistant” cells remained. In these resistant populations, we observed increased MET and EGFR colocalization and increased MET and EGFR activation. To understand how colocalization of MET and EGFR promotes signaling redundancy and crosstalk, we asked whether MET and EGFR directly interact in TNBC PDX using a proximity ligation assay (PLA). PLA uses antibody specificity to detect direct protein-protein interactions at physiologic levels in vitro and in vivo. We observed a significant increase in MET EGFR interactions only in resistant cell populations that had been treated with both MET and EGFR inhibitors. AKT and ERK signaling were also increased in TKI-resistant cells, indicating that these are key survival pathways in resistance. Interestingly, we did not observe an increase in MET-EGFR activity or interaction in PDX tumors treated for only 3 days. Even though TNBC cells (HCC70) showed increased colocalization of MET and EGFR with combined glesatinib and erlotinib treatment, there was not a significant increase in MET-EGFR interactions. Evaluation of MET-EGFR interaction in primary TNBC patient samples revealed significant MET-EGFR interactions in a MET-amplified tumor and lymph node metastases. To our knowledge, this is the first time MET-EGFR interactions have been observed in TNBC in vivo and in human patients. Moreover, MET-EGFR interactions are able to maintain MET, EGFR, ERK, and AKT activity through a yet-unknown mechanism. These results imply that MET EGFR interaction may be a unique mechanism of resistance to kinase inhibition that may be inherent in human TNBCs with genomic amplification of MET or EGFR. Citation Format: Elizabeth Tovar, Curt Essenburg, Anderson Peck, Lisa Turner, Zachary Madaj, Matthew Smith, James Christensen, Marianne Melnik, Eric Haura, Matthew Steensma, Carrie Graveel. MET and EGFR interaction promotes acquired resistance to kinase inhibition in TNBC [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A48.

Role of osteopontin as a predictive biomarker for anti-EGFR therapy in triple-negative breast cancer

ABSTRACTObjective: Effective targeted therapies for patients with triple-negative breast cancer (TNBC) present an unmet clinical need. There is evidence that TNBCs often have increased expression of the epidermal growth factor receptor (EGFR) and of osteopontin (OPN). OPN-mediated signaling can activate EGFR-dependent signaling pathways. Here, we assessed OPN as a potential predictive biomarker for response to anti-EGFR therapy in TNBC.Research design and methods: Using two different TNBC cell lines, MDA-MB-468 and MDA-MB-231, we investigated the impact of stable expression of OPN on efficacy of the EGFR inhibitor erlotinib in vitro.Results: We observed that breast cancer cells engineered to overexpress OPN are more sensitive to growth inhibition by erlotinib than control cells. The level of response was related to the level of OPN expression, possibly due to increased phosphorylation status of EGFR Tyr1068.Conclusions: These results indicate that OPN expression levels are related to sensitivity of TNBC cells to growth inhibition by erlotinib. OPN thus is a promising predictive biomarker for anti-EGFR therapy in breast cancer.

Fn14 deficiency ameliorates psoriasis-like skin disease in a murine model

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that acts through its receptor fibroblast growth factor-inducible 14 (Fn14). Recent studies demonstrated that the TWEAK/Fn14 signals participate in the development of psoriasis. The purpose of this study was to further explore the effect of Fn14 inhibition on experimental psoriasis. Psoriasis-like skin disease was induced in the wild-type and Fn14-knockout BALB/c mice. We found that Fn14 deficiency ameliorates psoriasis-like lesion in this model, accompanied by less inflammatory cell infiltration and proinflammatory cytokine production in lesional skin. The cutaneous expression of TNF receptor type 2 also decreased in the Fn14-deficient mice. Moreover, the topical application of TWEAK exacerbated psoriatic lesion in the wild-type but not in the Fn14-deficient mice. Furthermore, TWEAK promoted the expression of interleukin 8, keratin 17, and epidermal growth factor receptor (EGFR) but inhibited the expression of involucrin in psoriatic keratinocytes in vitro. Interestingly, such effect of TWEAK was abrogated by an EGFR inhibitor (erlotinib). TWEAK also enhances the proliferation and interleukin-6 production of dermal microvascular endothelial cells under psoriatic condition. In conclusion, TWEAK/Fn14 signals contribute to the development of psoriasis, and involves the modulation of resident cells and the transduction of the EGFR pathway. Fn14 inhibition might be a novel therapeutic strategy for patients with psoriasis.

Abstract 4452: Musashi-2 regulates EGFR/HER3 expression in NSCLC, cell proliferation and response to EGFR inhibitors in EGFR-mutant NSCLC

Abstract Musashi-2 (MSI2) is an RNA-binding protein that regulates mRNA translation. We recently established that MSI2 is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression and drives NSCLC metastasis, in part based on activity supporting a TGF-beta/SMAD3/claudin signaling cascade. Here, reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+;P53R172HΔG/+ murine NSCLC cell lines identified a significant ~2.7-fold upregulation of HER3 (ERBB3) upon MSI2 depletion. Negative MSI2-dependent regulation of ERBB3 protein was confirmed in multiple NSCLC models, based on analysis of MSI2 depletion or overexpression. Further, MSI2 positively regulated expression of the epidermal growth factor receptor (EGFR) protein in the same models. Comparing EGFR and KRAS driven models, we found that MSI2 depletion significantly impairs cell proliferation only in EGFR-mutant NSCLC cell lines. Using RNA immunoprecipitation analysis coupled with qPCR, we show that MSI2 directly binds to EGFR and, to a lesser extent, to HER3 mRNA. MSI2 mRNA binding was approximately correlating with the presence of predicted MSI2 binding sites in corresponding mRNAs. NSCLC lung tissue microarray analysis revealed that MSI2 total positivity by H-score, 2+/3+ and 3+ positivity correlated with EGFR 3+ staining. Finally, EGFR inhibitors erlotinib and afatinib synergized with MSI2 depletion in EGFR mutant models, suggesting that therapeutic targeting of MSI2 could be of clinical value, especially in EGFR-mutant lung cancer. Citation Format: Peter Makhov, Alexander Kudinov, Alexander Deneka, Brian L. Egleston, Emmanuelle Nicolas, Kathy Q. Cai, Rohan Brebion, Eleanor Avril, Mark Hitrik, Anna S. Nikonova, Ilya G. Serebriiskii, Vladimir Khazak, Hossein Borghaei, Erica A. Golemis, Yanis Boumber. Musashi-2 regulates EGFR/HER3 expression in NSCLC, cell proliferation and response to EGFR inhibitors in EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4452.

Abstract 4935: High-throughput immune-oncology screen identifies EGFR inhibitors as potent enhancers of CTL antigen-specific tumor cell killing

Abstract As immune checkpoint blocking antibodies increasing become foundational therapies for the treatment of cancer, there is a pressing need to identify compounds that synergize with checkpoint blockade as the basis of combinatorial treatment regimens. We have developed a screening assay in which a luciferized tumor cell line expressing a model antigen is co-cultured with a transgenic CD8+ T cell specifically recognizing the model antigen in a H-2b-restricted manner. The target tumor cell/T cell assay was screened with a small molecule library to identify compounds that inhibit or enhance T cell-mediated killing of tumor cells in an antigen-dependent manner. The EGFR inhibitor Erlotinib was the top hit that enhanced T cell killing of tumor cells. Subsequent experiments with Erlotinib and additional EGFR inhibitors validated the screen result. EGFR inhibitors increase both basal and IFN-γ-induced antigen processing and presentation of MHC class-I, which enhanced recognition and lysis by CD8+ cytotoxic T lymphocytes. The tumor cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen utilizing the same target tumor cell/T cell assay identified sgRNAs targeting EGFR as sensitizing tumor cells to T cell-mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in the MC38 syngeneic colon cancer model that was superior to PD-1 blockade or EGFR inhibition alone, further validating EGFR inhibitors as immunomodulatory agents that enhance PD-1 checkpoint blockade. This novel target tumor cell/T cell assay can be screened in high-throughput with small molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds AND target genes, respectively, that enhance or inhibit T cell recognition and killing of tumor cells. Citation Format: Patrick H. Lizotte, Troy Luster, Megan E. Cavanaugh, Luke J. Taus, Abha Dhaneshwar, Naomi Mayman, Aaron Yang, Mark Bittinger, Paul Kirschmeier, Nathanael S. Gray, David A. Barbie, Pasi A. Janne. High-throughput immune-oncology screen identifies EGFR inhibitors as potent enhancers of CTL antigen-specific tumor cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4935.

Abstract 826: Th1 cytokines and EGFR inhibition: A combinatorial therapeutic strategy in TNBC

Abstract Oncodrivers are a promising target in novel breast cancer therapy development, owing to oncogene addiction of breast cancer cells to sustain their malignancy. We have previously shown progressive loss of CD4+ T-helper type 1 response to oncodrivers in triple negative breast cancer (TNBC) patients. TNBC is marked by lack of cell surface ER, PR and Her2 receptors and is the most aggressive subtype of breast cancer. Lack of therapeutic targets, resistance to existing hormone therapy, metastasis and poor survival in TNBC patients underline the need for novel TNBC therapy development. In this study, we investigated the effect of EGFR inhibitors (cetuximab, erlotinib and lapatinib) alone or combination with Th1 cytokines (IFN-γ and TNF-α) on growth and proliferation of MDA-MB-231 and MDA-MB-468 TNBC cells. We observed decrease in EGFR expression in TNBC cells when treated with IFN-γ alone and in combination with EGFR inhibitors. In addition, IFN-γ alone, in combination with TNF-α and all three EGFR inhibitors, markedly increased STAT1 phosphorylation, indicating suppression of growth and proliferation in TNBC cells. Decreased STAT3 phosphorylation by combination treatment in TNBC cells may induce apoptosis and inhibit proliferation further. Combination of cetuximab and IFN-γ increased cleaved caspase-3 expression in MDA-MB-231, but not in MDA-MB-468 TNBC cells. EGFR inhibition and Th1 cytokine treatment showed combination treatment resulted in severe cell loss and morphological alteration, while Th1 cytokines alone did not induce significant senescence in MDA-MB-231 cells in senescence associated β-galactosidase assay. We are currently investigating activation status of other signaling pathways in TNBC cells, following Th1 cytokines treatment with EGFR inhibition. Our data suggests a combinatorial treatment approach, including DC1 vaccination to elicit Th1 immune response and inhibition of EGFR oncodriver, may lead to an effective and novel therapeutic strategy for triple negative breast cancer. Citation Format: Amrita Basu, Krithika Kodumudi, Doris Wiener, Brian Czerniecki. Th1 cytokines and EGFR inhibition: A combinatorial therapeutic strategy in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 826.

Abstract 1178: Genotype-fitness maps guide targeted therapy combination in lung adenocarcinoma

Abstract Targeted EGFR inhibition in lung cancer leads to dramatic responses. Nonetheless, disease evolution to resistance is the rule. As this evolutionary process is fueled by intra-tumoral genetic diversity, a comprehensive mapping of clonal fitness is required to inform strategies to overcome resistance. To model intra-tumoral diversity in vitro, we performed a genome-wide, over-expression genetic perturbation assay in EGFR-driven non-small cell lung cancer (NSCLC) PC9 cells. Our screen covered 17,255 ORFs (open reading frame constructs) representing 12,728 wildtype and mutated genes, and examined the effects of a first-generation EGFR inhibitor (erlotinib), a third-generation EGFR inhibitor (osimertinib) and a MEK inhibitor (binimetinib) on evolutionary selection, alone or in combination. Specifically, to obtain genotype-to-fitness maps for each drug, we measured clonal abundance up to 4 times during the screen and mathematically resolved their growth behavior. Finally, for each drug, we applied multiple doses to obtain dose-fitness relationships, which may impact tumor evolution in patients due to high inter- and intra-tumoral variability in drug delivery. In total, we have performed 78 genome-wide screens to map the evolutionary landscape of PC9 resistance to targeted therapy. Erlotinib and osimertinib both result in an overwhelming reduction of fitness, as expected from their clinical benefit. Known clinical resistance mechanisms involving ERBB2, PIK3CA, AXL, and BRAF confer a pronounced fitness advantage in the presence of both drugs, while EGFR (T790M) improves fitness only with erlotinib. Novel resistance mechanisms include alternative tyrosine kinases (NTRK, PDGFRB, CSF1R, KIT), KRAS, G-coupled protein receptors, transcription factors (SOX15, FOXA1), and cellular transporters (ABCG2). Notably, we observe significant divergence in dose-fitness relationships between different resistance mechanisms. For example, PIK3CA confers a modest but persistent advantage across the dose range, in contrast to BRAF which results in a fitness advantage only when sufficiently high drug levels are added. The fitness landscape for binimetinib resistance appeared to be largely orthogonal to that of the EGFR inhibitors, with decreased fitness noted for many tyrosine kinases as well as KRAS mutations. MEK inhibitor resistance results from RAF family member overexpression (ARAF, BRAF, RAF1) and MAPK activation. The orthogonal resistance landscapes of EGFR inhibition and MEK inhibition translate into highly synergistic effects in drug combination, with effective prediction of combinatorial fitness from the single-agent fitness landscapes using generalized linear models. These results validate this approach as a systematic method to address the combinatorial problem of optimizing drug combinations and doses to directly anticipate and address cancer evolution. Citation Format: Asaf Zviran, Patrick Bolan, Lisa Brenan, Amy Goodale, Denisse Rotem, Viktor Adalsteinsson, Federica Piccioni, Cory Johannessen, Dan Landau. Genotype-fitness maps guide targeted therapy combination in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1178.

Abstract 3897: Pharmacogenomic landscape of long non-coding RNAs in human cancers

Abstract Extensive efforts in recent large-scale drug screens have enabled identification of protein-coding genes (PCGs) as biomarkers for anti-cancer agent sensitivity, but information on the role of long-non coding RNAs (lncRNAs) in drug response is sparse. Lower abundance, difficulty in expression profiling and limited functional knowledge have so far hindered global analysis of lncRNAs in drug screens. However, lncRNAs represent nearly 60% of the human genome (vs. 3% for PCGs) and contain a vast majority of pharmacogenomic SNPs. Thus, it is critically important to evaluate the role of lncRNAs in drug sensitivity. Here, we perform a comprehensive reanalysis of large-scale drug screens using novel computational methods to create a landscape of pharmacogenomic interactions of lncRNAs in human cancers. We developed an algorithm to accurately impute the lncRNA transcriptome using PCG expression. This approach allowed us to generate lncRNA transcriptome in high throughput drug screening data sets and enabled systematic evaluation of lncRNAs for hundreds of drugs. For example, we applied our method to the Genomics of Drug Sensitivity in Cancer (GDSC) study, where sensitivity to 265 anti-cancer agents were quantified in 963 cancer cell lines. Sparse regression analysis showed lncRNA transcriptome were similar in accuracy at predicting drug response compared to PCGs (P = 0.9), indicating lncRNAs may be equally important biomarkers. Subsequently, we identified drug-specific lncRNA biomarkers using ANOVA while adjusting for tissue type. Our analysis recapitulated literature-verified lncRNAs associated with drug response, e.g. HOTAIR expression with cisplatin resistance (FDR &amp;lt; 0.05). In addition, we identified several compelling novel lncRNA associations for specific drugs along with previously uncharacterized multi-drug response predictors. We next identified lncRNAs that may serve as biomarkers for drug response while controlling for known cancer functional events. As an example, we analyzed EGFR-inhibitors (erlotinib, gefitinib, pelitinib) while controlling for EGFR somatic point mutations and amplification status and identified an anti-sense lncRNA overlapping UGT1A locus that may be a potential regulator of erlotinib metabolism. These findings reveal important lncRNAs that predict response independent of known cancer functional events and are currently being evaluated in non-small cell lung cancer patients. In conclusion, our comprehensive analysis has generated unprecedented insights into the role of lncRNAs in anti-cancer drug response and will be an invaluable resource for researchers studying drug response in various cancer types. Citation Format: Aritro Nath, R. Stephanie Huang. Pharmacogenomic landscape of long non-coding RNAs in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3897.

Abstract 1170: NCI-H1975 L858R/T790M/C797S isogenic cell line for next-generation EGFR inhibitor screening

Abstract Non-small-cell lung cancer (NSCLC) is the most common cause of cancer death. Over-activation of EGFR occurs in 13%~23% of NSCLC. The identification of EGFR as a cancer driver gene had led to rapid development of NSCLC targeted therapy, but the mutation of EGFR during treatment can cause drug resistance and cancer relapse. For example, T790M mutation causes resistance to the first generation of EGFR inhibitor Erlotinib and C797S mutation contributes to the resistance to the 3rd EGFR inhibitor AZD9291. There are several human cell lines for T790M mutation research, but there is not yet any primary human cancer cell line with C797S mutation. In the current study, a human isogenic EGFR C797S mutation cell line was generated through introducing nucleotide mutation into the EGFR gene in NCI-H1975 cell line using CRISRP technology, which originally bears L858R and T790M mutations of EGFR. First, the sgRNA sequence around the exon-20 of EGFR gene was screened using CRIPSR plasmid. Second, the donor plasmid with the homology arm containing exon-20 of EGFR and selecting marker was constructed and co-transfected into the parental cell line. Then, the NCI-H1975 C797S isogenic cell line was obtained and confirmed after screening over 100 single clones using the PCR method. The NCI-H1975 C797S isogenic cell line was resistant to AZD9291 but sensitive with EAI045 (the potential 4th generation EGFR inhibitor) combined with cetuximab. These data indicate that our NCI-H1975 L858R/T790M/C797S isogenic line is a useful model for next generation EGFR inhibitor study. Citation Format: Feng Hao, Feng He, Wenna Zhang, Zhaoshuai Bai, Jinying Ning. NCI-H1975 L858R/T790M/C797S isogenic cell line for next-generation EGFR inhibitor screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1170.

Abstract 832: An AXL-specific antibody-drug conjugate shows preclinical anti-tumor activity in non-small cell lung cancer, including EGFR-inhibitor resistant NSCLC

Abstract Enhanced AXL expression has been observed in tumor tissues obtained from non-small cell lung (NSCLC) patients, both in drug-treatment naïve patients and in patients with acquired resistance to EGFR tyrosine kinase inhibitors (EGFRi). In this study, we have evaluated the antitumor activity of the novel AXL-targeting antibody-drug conjugate (ADC) AXL-107-MMAE (HuMax-AXL-ADC) in non-small cell lung cancer (NSCLC) in vitro and in vivo. The antitumor activity of AXL-107-MMAE (4 mg/kg) was evaluated in vivo in a mouse patient-derived xenograft (PDX) clinical trial (1 mouse per group), using a collection of 57 NSCLC-derived PDX models, encompassing the typical NSCLC histological subtypes and mutational genotypes. AXL-107-MMAE induced responses, defined by a decrease in relative tumor growth compared to an untreated control tumor, in 35/57 (61%) of PDX models. Potent anti-tumor activity, i.e. tumor stasis or tumor regression, was observed in 16 out of 57 (28%) models. Potent anti-tumor activity was associated with higher AXL RNA expression compared to models showing intermediate response or non-responders (p&amp;lt;0.001). The therapeutic activity of AXL-107-MMAE (2 and 4 mg/kg) was confirmed in a panel of NSCLC PDX or cell line-derived xenograft models using 6-8 mice per group. AXL-107-MMAE induced dose-dependent, single agent anti-tumor activity in 8 out of 9 models. These included 2 EGFR-mutant PDX models that were resistant to the EGFR inhibitor erlotinib, including one model containing L858R/T790M EGFR mutations and one model with a L858R EGFR mutation. To specifically evaluate opportunities for AXL-107-MMAE in EGFR-mutant, treatment-resistant NSCLC, we established a panel of EGFR-mutant NSCLC cell lines with acquired resistance to the EGFRi erlotinib, gefitinib, or osimertinib. In general, cell lines showed enhanced AXL protein expression upon acquiring resistance to EGFRi. Moreover, the AXL-expressing, EGFRi-resistant cell lines were efficiently killed by AXL-107-MMAE in vitro, whereas the parental, EGFR-TKI sensitive NSCLC cell lines, which expressed little or no AXL, did not respond to treatment with AXL-107-MMAE. In summary, we show that AXL-107-MMAE has therapeutic activity as a single agent in the majority of NSCLC-derived models evaluated in vivo, representing different NSCLC histological and mutational subtypes. Moreover, AXL-107-MMAE induced efficient cytotoxicity in NSCLC cell lines that showed enhanced AXL expression upon acquired resistance to EGFRi. Finally, AXL-107-MMAE induces anti-tumor activity in EGFR-mutant, EGFRi resistant NSCLC PDX models. Citation Format: Louise A. Koopman, Maarten L. Janmaat, Kirstine Jacobsen, Mikkel Green Terp, Elke Gresnigt-van den Heuvel, Ulf Forssman, Andreas Lingnau, Paul W. Parren, Henrik Ditzel, Esther C. Breij. An AXL-specific antibody-drug conjugate shows preclinical anti-tumor activity in non-small cell lung cancer, including EGFR-inhibitor resistant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 832.