FGFR Pathway Research Articles

PO-163 Aerobic exercise activates myocardial FGF21/FGFR1/PI3K-AKT signaling pathway and inhibits cardiomyocyte apoptosis in post-myocardial infarction rats

Objective To investigate the effect of aerobic exercise on the expression of fibroblast growth factor 21 (FGF21) and cardiomyocyte apoptosis in Myocardial Infarction (MI) rats.
 Methods male SD rats were randomly divided into three groups：the sham operation (S), sedentary MI group (MI) and MI with aerobic exercise group (ME). The MI model was established by ligation of the left anterior descending branch of the left coronary artery. ME group were trained four weeks after the operation. LVSP, LVEDP and ±dp/dtmax were used to evaluate cardiac function. H9C2 cardiomyocytes were stimulated by 400 μmol/L H2O2 for 4h to simulate myocardial apoptosis mode. AMPK agonist AICAR and FGF21 receptor inhibitor PD166866 were used to interfere with H9C2. Myocardial collagen volume fraction was calculated by Masson staining and myocardium FGF21, FGFR1, Bax, Bcl-2 and PI3K-AKT pathway by western blotting or RT-Qpcr. Cardiomyocytes apoptosis was evaluated by TUNEL.
 Results Compared with S, the expression of FGF21, FGFR1, Bax, Bcl-2 and PI3K, AKT increased significantly in MI group, the apoptotic cardiomyocytes and collagen fibers increased significantly, but the cardiac function decreased. Compared to MI, myocardium FGF21, FGFR1 and PI3K, AKT were further increased in ME group, the Bax/Bcl-2 and the apoptotic cardiomyocytes decreased significantly. The percentage of collagen fibers decreased and the cardiac function was improved. Myocardium FGF21 was positively correlated with the inhibition of cardiomyocyte apoptosis and the improvement of cardiac function. Furthermore, the expression of Bax/Bcl-2, TNF-α/IL-10 and the apoptotic cardiomyocytes was significantly increased by PD166866, but PI3K-AKT pathway decreased significantly by PD166866. However, AICAR single intervention or PD166866 simultaneous intervention also can reverse this adverse effects.
 Conclusions Exercise can increase myocardial FGF21/FGFR1 with MI. The one of the mechanisms is to activate PI3K-AKT pathway to inhibit cardiaomyocyte apoptosis and inflammatory. It indicates that FGF21/FGFR1/PI3K-AKT signaling pathway plays an important role in inhibiting myocardial apoptosis and improving cardiac function.

MS32.04 Molecular Phenotypes of SCLC

Heterogeneity of neuroendocrine (NE) differentiation in small cell lung cancer (SCLC) determines very different molecular phenotypes Background and Purpose: SCLC is usually regarded as a homogenous tumor treated in a standard fashion. We have established and validated a 50 gene expression quantitative scoring system for NE lung tumors, consisting of both positive and negative scores [1]. While almost all carcinoid tumors have positive scores, SCLC and large cell neuroendocrine carcinomas have positive and negative subpopulations. Methodology: We utilized the NE gene expression score to demonstrate intertumor heterogeneity of gene expression patterns and their relationship to signaling pathways and to immune and inflammatory response. We used cluster analysis to divide the SCLC samples into high and low NE subtypes. We studied two public SCLC tumor data sets (n = 140) as well as 70 cell lines established by us and a pair of high/low sublines established from a single SCLC cell line. The NE score cluster analysis indicated that ∼10% of cell lines and ∼20 of SCLC tumors were of the low NE subtype. The low NE subtype samples had RB1 mutations or low or absent RB1 expression, confirming their SCLC origin. Differences between the high and low subtypes were divided into those common to tumors and cell lines and those largely or entirely limited to tumors. Differences common to tumors and cell lines: At least three different lineage specific transcription factors were expressed by SCLC tumors and cell lines. Most NE high cells expressed ASCL1 in conjunction with NKX2-1. NEUROD1, occasionally with ASCL1, was expressed by a smaller subset of NE high and occasionally by NE low cells. POU2F3 was expressed by most of the NE low subtypes, and about half of these also expressed ASCL2. However, about 4% of tumors and cell lines (all belonging to the NE low subtype) lacked an identified lineage specific transcription factor. The NE high subtype expressed all of the classic NE markers, as well as numerous neural markers covering neural and glial differentiation. In addition they selectively expressed markers for DNA damage, the FGFR pathway, calcium signaling and Notch inhibitors. The NE low subtype lacked all or most of the NE cell program, NKX2-1 and DLL3 expression, while MYC and REST expression, the Hippo, TGFb and Notch pathways and EMT were greatly upregulated. These and other findings were predicted to impact on the potential responses of the two subtypes to chemotherapy, radiotherapy, and multiple targeted therapies. Differences limited to tumors and absent in cell lines: Despite having a high mutational burden, SCLC is considered to be an “immunological desert”, with few infiltrating immune or inflammatory cells, low or absent PDL-1 expression, and modest responses to PD-1 blockade. While the high NE tumors fitted this description most of the low NE tumors had strands of fibrosis encircling tumor islets, with varying numbers of inflammatory cells in the fibrous strands. Major differences at multiple steps were present in the low NE subtype involving the over 200 genes regulating the Cancer-Immunity Cycle, including the major cytokine families, toll like receptors, the inflammasome pathway, the JAK/STAT pathway, HLA antigens, CD47 antigen, perforin and granzymes. These changes were accompanied by highly significant increases in the relative numbers of multiple types of infiltrating immune and inflammatory cells. In addition, the low NE subtype had increased expression of PDL-1 and the interferon gamma signature, suggesting enhanced responses to immunotherapy. SCLC tumors and cell lines can be divided into high and low NE subtypes and these subtypes have major differences in lineage specific transcription factors, expression of the NE program, as well as some oncogenes and pathways, predicting them to respond very differently to conventional and targeted therapies. In addition, very different infiltrating cellular and gene expression patterns of immune responses were present in the low NE tumors, involving multiple aspects of Innate and Adaptive Immunity, and predicting that the low NE subtype tumors are more likely to respond to immunotherapy. In addition, several possibilities of combining cytotoxic, targeted and immunotherapies are suggested from our findings. I thank my collaborators for their invaluable contributions: Ling Cai, Tao Wang, Guanghua Xiao, Luc Girard and John Minna. Reference: 1. Zhang W, Girard L, Zhang YA, et al. Small cell lung cancer tumors and preclinical models display heterogeneity of neuroendocrine phenotypes. Transl Lung Cancer Res 2018;7(1):32-49.

Everolimus and pazopanib (E/P) benefit genomically selected patients with metastatic urothelial carcinoma

BackgroundMetastatic urothelial carcinoma (mUC) is a genomically diverse disease with known alterations in the mTOR pathway and tyrosine kinases including FGFR. We investigated the efficacy and safety of combination treatment with everolimus and pazopanib (E/P) in genomically profiled patients with mUC.MethodsmUC patients enrolled on a Phase I dose escalation study and an expansion cohort treated with E/P were included. The primary end point was objective response rate (ORR); secondary end points were safety, duration of response (DOR), progression-free survival (PFS) and overall survival (OS). Patients were assessed for mutations and copy number alterations in 300 relevant cancer-associated genes using next-generation sequencing and findings were correlated with outcomes. Time-to-event data were estimated with Kaplan–Meier methods.ResultsOf the 23 patients enrolled overall, 19 had mUC. ORR was 21% (one complete response (CR), three partial responses (PR), eight with stable disease (SD). DOR, PFS and OS were 6.5, 3.6, and 9.1 months, respectively. Four patients with clinical benefit (one CR, two PR, one SD) had mutations in TSC1/TSC2 or mTOR and a 5th patient with PR had a FGFR3–TACC3 fusion.ConclusionsCombination therapy with E/P is safe in mUC and select patients with alterations in mTOR or FGFR pathways derive significant clinical benefit.

FGFR1:TACC1 fusion is a frequent event in molecularly defined extraventricular neurocytoma.

Extraventricular neurocytoma (EVN) is a rare primary brain tumor occurring in brain parenchyma outside the ventricular system. Histopathological characteristics resemble those of central neurocytoma but exhibit a wider morphologic spectrum. Accurate diagnosis of these histologically heterogeneous tumors is often challenging because of the overlapping morphological features and the lack of defining molecular markers. Here, we explored the molecular landscape of 40 tumors diagnosed histologically as EVN by investigating copy number profiles and DNA methylation array data. DNA methylation profiles were compared with those of relevant differential diagnoses of EVN and with a broader spectrum of diverse brain tumor entities. Based on this, our tumor cohort segregated into different groups. While a large fraction (n = 22) formed a separate epigenetic group clearly distinct from established DNA methylation profiles of other entities, a subset (n = 14) of histologically diagnosed EVN grouped with clusters of other defined entities. Three cases formed a small group close to but separated from the epigenetically distinct EVN cases, and one sample clustered with non-neoplastic brain tissue. Four additional samples originally diagnosed otherwise were found to molecularly resemble EVN. Thus, our results highlight a distinct DNA methylation pattern for the majority of tumors diagnosed as EVN, but also indicate that approximately onethird of morphological diagnoses of EVN epigenetically correspond to other brain tumor entities. Copy number analysis and confirmation through RNA sequencing revealed FGFR1-TACC1 fusion as a distinctive, recurrent feature within the EVN methylation group (60%), in addition to a small number of other FGFR rearrangements (13%). In conclusion, our data demonstrate a specific epigenetic signature of EVN suitable for characterization of these tumors as a molecularly distinct entity, and reveal a high frequency of potentially druggable FGFR pathway activation in this tumor group.

Abstract 964: Genome-wide CRISPR screen identifies potential therapeutic combination of EGFR and FGFR inhibitors in oral cancer

Abstract Oral squamous cell carcinoma (OSCC) has remained a disease with poor survival for decades. Current treatment options such as targeting EGFR has had limited success. We hypothesize that inhibiting compensatory pathways in combination with EGFR signaling may result in greater efficacy. To identify synergistic combinations with EGFR-targeted therapy, we introduced Genome-scale CRISPR-Cas9 KnockOut (GeCKO) libraries into OSCC cell lines. CRISPR-Cas9 generates individual genetic knockouts through targeted gene editing. We used a pool of CRISPRs targeting over 18,000 genes to perform genome-scale screening for drivers of sensitivity to EGFR-targeted therapy. Upon selection of the OSCC GeCKO pool, we identified gene knockouts in the FGFR pathway that increased sensitivity to the EGFR inhibitor gefitinib. Using resazurin viability assays we tested combinations of EGFR and FGFR inhibitors in 14 OSCC cell lines. Six/14 (43%) of the cell lines were responsive to the combination, indicating that the FGFR pathway is an alternate mechanism of resistance to EGFR-targeted therapy in some tumors. We advanced an OSCC cell line to test in vivo by establishing subcutaneous cell line xenografts in mice. After treatment for 21 days, the mice receiving the combination of EGFR and FGFR inhibitors, gefitinib and BGJ398, had significantly smaller tumors than mice receiving vehicle or either monotherapy. These results suggest targeting EGFR in combination with its compensatory pathway such as FGFR signaling could be effective in vivo. While OSCC remains a common and frequently lethal cancer, there is great potential for the development of novel personalized targeted therapies. Here, we describe the use of genome-wide CRISPR-Cas9 library to discover the synergistic combination of EGFR and FGFR inhibition. Further investigation suggests the FGFR pathway is a common compensatory mechanism to EGFR inhibition, and that targeting EGFR and FGFR in combination has efficacy in an in vivo model. Overall, targeting compensatory mechanisms in combination with EGFR-targeted therapy could generate novel, beneficial treatment plans for patients with OSCC. Citation Format: Megan Ludwig, Andrew Birkeland, Sai Nimmagadda, Sue Foltin, Aditi Kulkarni, Hui Jiang, Thomas Carey, John Chad Brenner. Genome-wide CRISPR screen identifies potential therapeutic combination of EGFR and FGFR inhibitors in oral cancer [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 964.

Multicenter phase 2 study of nintedanib in patients (pts) with advanced progressing carcinoid tumors.

4105Background: Serotonin is the cause of carcinoid symptoms and can signal the formation of fibroblasts via fibroblast growth receptors (FGFR). Nintedanib is an oral inhibitor of the FGFR pathway ...

Abstract PD4-11: Copy-number and targeted sequencing analyses to identify distinct prognostic groups: Implications for patient selection to targeted therapies amongst anti-endocrine therapy resistant early breast cancers

Abstract Hormone receptor positive breast cancer remains an ongoing therapeutic challenge. Despite optimal anti-endocrine therapies, most breast cancer deaths follow a diagnosis of early luminal cancer. Data describing molecular events in breast cancer has yet to be translated into actionable information to inform medical management and benefit patients. To understand the impact of multiple aberrations in the context of current therapy, we assessed the prognostic ability of genomic signatures as a putative stratification tool to targeted therapies. The analysis was performed based on an a priori hypothesis relating to molecular pathways which might predict response to targeted therapies currently under evaluation in late-stage clinical trials. In a case-control fashion, 420 patients from the Tamoxifen vs Exemstane Adjuvant Multinational Trial (TEAM) pathology cohort, were analysed to determine the prognostic, ability for these mutational and copy-number biomarkers representing the CCND/CDK, FGFR/FGF and AKT/PIK3CA to inform potential response to therapies targeting these pathways. Copy number analysis was performed using the Affymetrix Oncoscan™ Assay. Targeted sequencing was performed in a subset of samples for genes based on signaling cassettes mined from the ICGC. Pathways were identified as aberrant if there were copy number aberrations (CNAs) and/or mutations in any of the predetermined pathway genes: 1) CCND1/CCND2/CCND3/CDK4/CDK6 2) FGFR1/FGFR2/FGFR2/FGFR4 and 3) AKT1/AKT2/PIK3CA/PTEN. Kaplan Meier and log rank analyses were used for DRFS between groups. Hazard ratios were calculated using the Cox proportional hazard models adjusted for age, tumour size, grade, lymph node and HER2 status. 390/420 samples passed informatics QC filters. For the CCND/CDK pathway, patients with no CNA changes experienced a better DRFS (HR=1.94, 95% CI 1.45-2.61, p< 0.001). For the FGFR/FGF pathway, a similar outcome is seen among patients without CNAs (HR = 1.43, 95% CI 1.07-1.92 p=0.017). For AKT/PIK3CA, a decrease in DRFS was seen in those with aberrations (H=1.34, 95% CI 1.00-1.81, p=0.053). We demonstrated that CNAs of genes within CDK4/CCND, PIK3CA/AKT and FGFR pathways are independently linked to high risk of relapse following endocrine treatment. In this way, improving the clinical management of early breast cancers could be made, firstly by identifying those patients for whom current endocrine therapies are sufficient, thus reducing unnecessary treatment; and secondly, identifying those patients who are at high-risk for recurrence despite optimal endocrine therapy and the linking molecular features driving these cancers to treatment with targeted therapies. Citation Format: Bayani J, Kornaga EN, Crozier C, Jang GH, Kalatskaya I, Trinh QM, Yao CQ, Livingstone J, Hasenburg A, Kieback DG, Markopoulos C, Dirix L, Boutros PC, Spears M, Stein LD, Rea D, Bartlett JMS. Copy-number and targeted sequencing analyses to identify distinct prognostic groups: Implications for patient selection to targeted therapies amongst anti-endocrine therapy resistant early breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-11.

Abstract P5-21-20: Integrating comprehensive genomic profiling with treatment decisions – Experience gained while treating 139 advanced breast carcinomas

Abstract Background: Comprehensive Genomic Profiling (CGP) using next-generation sequencing (NGS) technology can provide insight into potentially clinically relevant genomic alterations (CRGA) within a patient's breast cancer. For example, HER2 amplification status and targetable short variants (SV), acquired ESR1 or BRCA1/2 resistance mutations, and the presence of targetable alterations in the PI3K kinase. We retrospectively reviewed CGP results and subsequent outcomes at one cancer center to illustrate the experience of using molecular subtyping to inform treatment decisions. Methods: DNA extracted from FFPE tumor tissue or blood samples obtained during routine clinical care for patients (n=139) with predominantly relapsed, refractory or metastatic breast cancer was analyzed by hybrid-capture, NGS for all classes of GA: 1. base substitutions, 2. insertion and deletions, 3. rearrangements, and 4. copy number changes. Treatment decisions based on comprehensive genomic profiles were captured retrospectively. Tumor mutational burden (TMB), scored as mutations (mut)/Mb, was calculated on 0.8-1.2 Mb of sequenced DNA. Alterations affecting the ERBB family included amplification of or oncogenic mutations in ERBB2 (HER2), ERBB3, and EGFR. Results: From Jan 2013 to May 2017, FFPE tissue samples for 136 patients with advanced breast cancer were analyzed by CGP and 3 additional patients had circulating tumor DNA analyzed for alterations; 11 patients received profiling on multiple biopsies. Tumors analyzed were carcinomas (Ca) NOS (n=84), invasive ductal Ca (n=46), invasive lobular Ca (n=7), a neuroendocrine Ca, and a phyllodes tumor. In total, 118/139 (84.9%) samples harbored CRGA in a targetable pathway: PI3K/MTOR (n=67; 48.2%), CDK cell-cycle (n=40; 28.8%), ERBB family (n=24; 17.3%), FGFR (n=24; 17.3%), ESR1 (n=16; 11.5%), homologous repair (HRD)( n=14; 10.1%), and RAS/RAF/MEK (n=11; 7.9%). Targetable alterations in other cancer-related kinases were found in 10 (7.2%) samples and 10 (7.2%) samples were TMB high (≥20 mut/Mb) or had CD274 (PD-L1) amplification. There were 3 patients (2.1%) with HER2 short variants detected in the absence of ERBB2 amplification; these patients may respond to HER2-targeted therapies but would be HER2-negative by IHC. Many samples had alterations in ≥1 pathway, and overlap is particularly high for the CDK and FGFR pathways (12 samples). Alterations in pathways targeted by MTOR inhibitors, HER2-targeted therapies, or the CDK inhibitors were found in 93/136 (66.9%) tumors. Evaluation of outcomes for these 139 patients is ongoing and will be presented. Conclusions: Genomic profiling of breast carcinomas, using either tissue or liquid biopsies, provides potentially actionable information to guide treatment decisions. Overall, 84.9% of patient samples harbored oncogenic alterations in a targetable pathway, with two-thirds of tumors having alterations in pathways targeted by therapies with FDA approval for breast cancer and 7.2% of patients having high levels of TMB or amplification of PD-L1, suggesting that checkpoint inhibitors may be relevant options. Citation Format: Mahtani R, Gay LM, Chung J, Hartmaier R, Sokol E, Elvin JA, Daniel S, Ramkissoon S, Severson E, Suh J, Vergilio J-A, Stephens PJ, Ross JS. Integrating comprehensive genomic profiling with treatment decisions – Experience gained while treating 139 advanced breast carcinomas [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-20.

Abstract GS6-05: Gain-of-function kinase library screen identifies FGFR1 amplification as a mechanism of resistance to antiestrogens and CDK4/6 inhibitors in ER+ breast cancer

Abstract Background: CDK4/6 inhibitors have been approved in combination with endocrine therapy for treatment of ER+ metastatic breast cancer. The goal of this study was to discover mechanisms of resistance to ER antagonists alone and in combination with CDK4/6 inhibitors. Results: To achieve this goal, we used lentiviral vectors to individually express 559 human kinase open reading frames (ORFs) in ER+ MCF7 human breast cancer cells treated with fulvestrant ± the CDK4/6 inhibitor ribociclib. In MCF7 cells treated with fulvestrant alone or with ribociclib, we identified 15 and 17 kinases associated with resistance, respectively. Ten of these kinases overlapped in both groups. In a secondary screen, MCF7 cells were stably transduced with V5-tagged lentiviruses expressing the positive 'hits' for treatment with fulvestrant/ribociclib. Five of 17 kinases (FGFR1, FRK, HCK, FGR, CRKL) were confirmed to induce resistance to fulvestrant/palbociclib and fulvestrant/ribociblib. Survey of TCGA for copy number alterations and/or expression of these 5 genes showed only FGFR1 to be amplified/overexpressed in ˜15% of ER+ breast cancers. Experiments in vitro showed that ER+/FGFR1-amplified (amp) MDA-134, CAMA-1 and HCC1500 human breast cancer cells and MCF7 cells stably transduced with FGFR1 were relatively resistant to estrogen deprivation, fulvestrant and fulvestrant/palbociclib compared to non-FGFR1 amp MCF7 cells. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Treatment with fulvestrant or palbociclib alone modestly delayed growth of ER+/FGFR1-amp breast cancer patient-derived xenografts (PDX) established in nude mice. However, addition of the FGFR TKI erdafitinib to fulvestrant/palbociclib resulted in marked PDX regression in all mice without associated toxicity and a complete cell cycle arrest measured by Ki67. Treatment of FGFR-amp cells with FGF-2 strongly induced CCND1 (cyclin D1) expression. Downregulation of CCND1 with CCND1 RNAi oligonucleotides restored sensitivity of FGFR1-amp cells to fulvestrant/palbociclib, thus phenocopying the effect of FGFR TKIs. Conversely, overexpression of CCND1 in MCF7 cells induced resistance to estrogen deprivation and to fulvestrant ± palbociclib. Finally, we examined next gen sequencing of cell free tumor DNA by Guardant360 in 34 patients before and after progression on CDK4/6 inhibitor. In 10/34 (29%) post-progression specimens, we detected alterations in the FGFR pathway: FGFR1 amplification (n=7), FGFR1 N546K (n=1), FGFR2 N549K (n=1), and FGFR2 V395D (n=1) activating mutations. Conclusions: These data suggest aberrant FGFR signaling is a mechanism of resistance to anti-ER therapies ± CDK4/6 inhibitors. We posit overexpression of cyclin D1 induced by both FGFR signaling and ER transcription plays a role in drug resistance. Based on these findings we propose ER+/FGFR1 amplified breast cancers are endocrine resistant and should be candidates for treatment with combinations of ER and FGFR antagonists. Accordingly, we have initiated a phase Ib trial of fulvestrant, palbociclib and erdafitinib in patients with antiestrogen resistant ER+/HER2-negative breast cancer with FGFR1-4 amplification. Citation Format: Formisano L, Lu Y, Jansen VM, Bauer JA, Hanker A, Gonzalez Ericsson P, Lee K-M, Nixon MJ, Guerrero-Zotano AL, Schwarz LJ, Sanders M, Sudhan D, Dugger TC, Cruz MR, Behdad A, Cristofanilli M, Bardia A, O'Shaughnessy J, Mayer IA, Arteaga CL. Gain-of-function kinase library screen identifies FGFR1 amplification as a mechanism of resistance to antiestrogens and CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS6-05.

Different stages in drug development for muscle-invasive bladder cancer.

Muscle-invasive bladder cancer (MIBC) is a highly aggressive disease. Despite optimal therapy, half of the patients will succumb to disease. This prognosis could not be improved over the last three decades. Therefore, MIBC is left behind from other cancers such as prostate, where novel treatment options were discovered and improve patient outcomes. While being aware of the recent emerging evidence of checkpoint inhibition in MIBC, we aim to describe different stages of drug development in MIBC by using three specific targets. On the example of Her2 targeting, we aimed to indicate, that either a target is ineffective in MIBC or that the patient selection is insufficient. The first clinical trials using a pan fibroblast growth factor receptor (panFGFR) inhibitor to target the FGFR pathway showed promising results. Data of further trials are to be awaited before implementing these drugs into daily clinical practice. A large variety of novel agents are investigated in vitro and in vivo. On the example of a malaria protein, we aimed to discuss a treatment paradigm that is not dependent on pathway signaling and the genomic landscape of MIBC. The ultimate question still remains to be answered: How do we select the optimal treatment for the right patient?

Abstract 54: Genome-wide CRISPR screen identifies potential therapeutic combination of EGFR and FGFR inhibitors in oral cancer

Abstract Oral squamous cell carcinoma (OSCC) is a disease with poor survival with few novel treatment options in development. While EGFR is commonly over-expressed, targeting EGFR has had limited success possibly because of compensatory pathways rescuing cell survival. Inhibiting compensatory pathways in combination with EGFR signaling may result in greater efficacy. To identify compensatory pathways, we used a Genome-wide CRISPR-Cas9 Knock-Out (GeCKO) library to identify genetic knockouts that sensitized OSCC cell lines to EGFR inhibition. This CRISPR library established a pool of knockouts targeting over 18,000 genes, and upon selection of the GeCKO pool we identified gene knockouts in the FGFR pathway increased sensitivity to the EGFR inhibitor gefitinib. We further profiled this combination with viability assays and found that 6/14 (43%) cell lines responded to the combination of EGFR and FGFR inhibition, suggesting that the FGFR pathway could be a common compensatory mechanism to loss of EGFR signaling. In complement, we again used CRISPR-Cas9 to generate a syngeneic EGFR knockout (KO) OSCC cell line. The parent OSCC cell line is responsive to the EGFR and FGFR dual inhibition, and the EGFR KO derivative retains sensitivity to FGFR-targeted monotherapies. We expect that this EGFR KO cell line will be a useful tool in further evaluating the compensatory mechanism of the FGFR pathway. In total, we describe the use of genome-wide CRISPR-Cas9 library to discover the synergistic combination of EGFR and FGFR inhibition, with follow-up indicating that the FGFR pathway could be a common compensatory mechanism to EGFR inhibition. Targeting compensatory mechanisms in combination with EGFR-targeted therapy could be clinically beneficial, including the combination of EGFR and FGFR inhibition. Citation Format: Megan Ludwig, Andrew Birkeland, Sai Nimmagadda, Sue Foltin, Aditi Kulkarni, Hui Jiang, Thomas Carey, J. Chad Brenner. Genome-wide CRISPR screen identifies potential therapeutic combination of EGFR and FGFR inhibitors in oral cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 54.

Abstract 52: Dynamic rewiring of apoptotic signaling networks by combined EGFR/FRGR inhibition enhances killing of head and neck squamous tumor by DNA damage

Abstract We have previously shown that a subset of triple-negative breast cancers can be dramatically sensitized to killing by DNA-damaging chemotherapy by time-staggered inhibition of EGFR. This sensitization effect results from dynamic re-wiring of apoptotic signaling pathways to engage a caspase-8-dependent cell death mechanism (Figure 1). Here we explored whether combined EGFR and/or FGFR inhibition could enhance the killing of head and neck squamous cell cancers by DNA damaging treatments through a similar re-wiring mechanism. The epidermal growth factor receptor (EGFR) and FGFR pathways are two of the most dysregulated molecular pathways in human head-and-neck squamous cell carcinoma (HNSCC). Despite overexpression in approximately 90% of HNSCC tumors, EGFR inhibitors alone have not exerted a major therapeutic impact on tumor response or patient survival. A recent study provides evidence that the insensitivity of the majority of HNSCC to EGFR inhibitors is mediated by dominant activity of alternative receptor tyrosine kinase (RTK) systems such as FGFR signaling (Clinical Cancer Research 2011). We have found that time-staggered administration of either EGFR inhibitors, or FGFR inhibitors, can dramatically enhance the apoptotic response of HNSCCs to DNA damage from doxorubicin. Importantly, combined inhibition of both the EGFR and FGFR pathways was more effective than inhibition of either pathway alone, at inducing sensitization to cytotoxic chemotherapy through dynamic network re-rewiring. Enhanced cell death by dual EGFR/FGFR inhibition and subsequent genotoxic injury results from greater activation of Caspase-8 than that seen by either EGFR or FGFR inhibitors alone. This enhanced Caspase-8 acitivty, together with Caspase-9 activation, accounts for the majority of apoptotic death. Our data further suggest that Src family kinases are a critical downstream node of EGFR/FGFR activity since treatment of HNSCC cell lines with Src kinase inhibitors resulted in similar sensitization to doxorubicin treatment. In summary, our data extend the utility of therapeutic dynamic rewiring for the targeting of tumors with redundant receptor signaling pathways as well as their critical downstream nodes. Citation Format: Yogesh Dayma, Michael B. Yaffe. Dynamic rewiring of apoptotic signaling networks by combined EGFR/FRGR inhibition enhances killing of head and neck squamous tumor by DNA damage [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 52.

Abstract IA25: Targeted therapies for hepatocellular carcinoma

Abstract In 2008, the multikinase inhibitor Sorafenib consistently proved to extend the survival of patients with Hepatocellular Carcinoma (HCC) that were treated in the advanced stage and had a good liver function, and it became the first agent to be specifically approved for this indication (ref). Although Sorafenib was a paradigm shift in the field, systemic therapy of HCC remains a major unmet need. The mechanism of action of Sorafenib is still poorly understood and the same is true for the mechanism of resistance after initial response, which usually reveals as disease stabilization for a few months. Molecular biomarkers predicting response and resistance to Sorafenib are also hardly defined. Furthermore, all subsequent novel targeted agents or regimens tested in large randomized controlled trials both in the first-line and second-line setting have been unable to reach or improve the magnitude of benefit obtained with Sorafenib. Strategies under study have included the blockade of other pro-angiogenic signals such as the FGFR pathway using Brivanib, proliferative signals such as the EGFR pathway using Erlotinib, or cell growth, proliferation, angiogenesis and apoptosis signals arising from the PI3K-Akt-mTOR pathway using Everolimus. The relevance of targeting these pathways is still not completely understood since biomarker data have not been reported, including EGFR and FGFR expression in tumor specimens. Besides, a number of trials have definitively shown the lack of efficacy of Doxorubicin, used for decades as a systemic therapy for HCC patients without formal scientific evidence. And no survival benefit was proven for combination chemotherapy based on 5-fluorouracil and oxaliplatin. The door was then opened to a more focused approach in which only patients with an altered targeted pathway are deemed candidates for a given agent. The anticancer activity of the c-met tyrosine kinase inhibitor Tivantinib was suggested in a randomized, placebo-controlled trial in the second-line setting where patients with high c-met expression survived significantly longer if they received Tivantinib. Patient enrichment based on molecular targets has been incorporated into the clinical development of other agents such as a monoclonal antibody that binds to glypican-3 or Refametinib a tyrosine kinase inhibitor that targets the MAPK (Ras-Raf-MEK-ERK) pathway. Agents targeting novel signaling pathways including TGF-β and FGF19, but also less explored pathways such as Wntβ-catenin, Hedgehog or Notch are being studied for the treatment of HCC. We will soon learn whether or not this more selective approach translates into positive results. Identification of driver molecular events in an individual patient using markers from tumor biopsies has proven successful in other tumors including breast or lung cancer. However, the reliability of this strategy has been challenged by studies showing intratumoural heterogeneity in various malignancies at advanced stages. HCC needs to be explored in this regard. Citation Format: Bruno Sangro. Targeted therapies for hepatocellular carcinoma [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA25.

PUB045 Anti-Tumor Effect of Honokiol in Lung Squamous Cell Carcinoma

Patients with lung squamous cell carcinoma (SCC) still benefit few from new therapies of lung cancer, and seeking for potential effective therapies is imperative. Honokiol (HNK) is a natural compound isolated from the magnolia plant with numerous pharmacological activities and exerts anticancer activity. Here, we demonstrated that the effect of HNK in lung SCC cells in vitro and related signaling mechanisms. Cell viability of human lung SCC cell lines NCI-H520 and SK-MES-1 exposed to HNK were measured by CCK-8. Flow cytometry was applied to identify the cellular apoptosis with HNK exposure of SCC cells compared to lung adenocarcinoma cells. Fibroblast growth factor(FGF)-2 expression of both SCC cells with HNK exposure were explored by quantitative real-time PCR. Furthermore, the effect of HNK on FGFR1 and signal pathways related to proliferation, apoptosis and cell cycle were detected by western blot. In two subpopulations of human lung SCC cells, NCI-H520 and SK-MES-1, HNK exposure remarkably inhibited the cell proliferation in both time-and dose-dependent manners. Compared to A549 cells, a more significant cell apoptosis was observed in NCI-H520 cells after HNK incubation. Next, real-time PCR analysis indicated that HNK down-regulated the expression of FGF2 in both NCI-H520 cells and SK-MES-1 cells in dose dependence. Meanwhile, HNK also obviously inhibited FGFR1 in both cell lines. Moreover, HNK exposure activated PARP and caused caspase-3 cleavage, suppressed cyclineD1 and ERK expression. These results broaden our understanding of the mechanisms on HNK effects in lung SCC, and reinforce the possibility of its potential anticancer benefit on SCC patients.

Nucleoside/nucleotide reverse transcriptase inhibitors attenuate angiogenesis and lymphangiogenesis by impairing receptor tyrosine kinases signalling in endothelial cells.

Cardiovascular disease associated with antiretroviral therapy (ART) has become a major clinical challenge for HIV-positive patients. However, the role of ART in blood vessel growth is largely unknown. Here, we examined an integral component of ART, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) and investigated their effects on key microvascular functions, including angiogenesis and lymphangiogenesis. The angiogenesis/lymphangiogenesis capability of endothelial cells (ECs) was evaluated using migration, proliferation and tube formation assays in vitro, and mouse ear and Matrigel plug assays in vivo. Expressions of signalling molecules and mitochondrial antioxidant catalases were determined using Western blotting. Receptor tyrosine kinase (RTK) internalization and endocytosis were examined using flow cytometry and confocal immunofluorescence microscopy respectively. Mitochondrial DNA copy number and ROS were determined using quantitative real-time PCR and MitoSOX staining respectively. Pharmaceutical doses of NRTIs [azidothymidine (AZT), tenofovir disoproxil fumarate (TDF) and lamivudine (3TC)] inhibited angiogenesis and lymphangiogenesis both in vivo and in vitro by affecting the proliferation and migration of ECs. Correspondingly, NRTIs selectively attenuated the activation and transduction of endothelial RTK signals, VEGFR2 and FGFR1 pathways, in vascular ECs and the VEGFR3 pathway in lymphatic ECs. Both TDF and 3TC restrained RTKs' endocytosis into early endosomes but not internalization, while AZT blocked the protein maturation of RTKs. Excessive ROS levels were detected in NRTI-treated ECs, and the MnSOD mimic MnTMPyP alleviated the angiogenic/lymphangiogenic defects induced by NRTIs. NRTIs negatively regulate angiogenesis and lymphangiogenesis by inducing mitochondrial oxidative stress and subsequently impairing RTK signalling in ECs. This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

Abstract B27: Identifying therapeutic combinations with EGFR-targeted therapy through the generation of genome-wide CRISPR-Cas9 knockout libraries in oral cancer cell lines

Abstract Oral squamous cell carcinoma (OSCC) remains a disease with poor survival. With recent characterization of the mutational landscape of OSCCs, there is great potential for personalized targeted therapies. However, to date utilization of targeted therapies in OSCC have had limited success with anti-EGFR therapy. Combinations of targeted therapies, which may have greater efficacy by inhibiting compensatory pathways, have not been well studied. To identify synergistic combinations with EGFR-targeted therapy, we introduced Genome-scale CRISPR-Cas9 Knock-Out (GeCKO) libraries into OSCC cell lines. CRISPR-Cas9 generates individual genetic knockouts through targeted gene editing, and by using a pool of CRISPRs targeting over 18,000 genes we can perform genome-scale screening for synergistic targetable pathways. Upon selection of the OSCC GeCKO pool, we identified that knockouts of genes in the FGFR pathway caused sensitivity to the EGFR inhibitor gefitinib. We further investigated the possible synergistic effect of dual EGFR and FGFR inhibition with resazurin viability assays in 14 of our OSCC models. We found 6/14 (43%) of the models were responsive to the combination of EGFR and FGFR inhibition, indicating that the FGFR pathway could be an alternate mechanism of resistance to EGFR-targeted therapy. For further investigation of this combination, we again used CRISPR-Cas9 to generate an EGFR knockout (KO) OSCC cell line. This EGFR KO cell line retains sensitivity to FGFR inhibitors, and we expect that this cell line will be a useful tool in further evaluating the compensatory mechanism of the FGFR pathway. OSCC remains a common and frequently lethal cancer with great potential for the development of personalized targeted therapies. Here, we describe the use of genome-wide CRISPR-Cas9 library to discover the synergistic combination of EGFR and FGFR inhibition. Further investigation suggests the FGFR pathway as a common compensatory mechanism to EGFR inhibition. We hope to use this approach to identify additional compensatory mechanisms of resistance to targeted therapies with the eventual goal of translating these findings to clinic. Citation Format: Megan Ludwig, Andrew Birkeland, Sai Nimmagadda, Sue Foltin, Hui Jiang, Thomas Carey, John Chad Brenner. Identifying therapeutic combinations with EGFR-targeted therapy through the generation of genome-wide CRISPR-Cas9 knockout libraries in oral cancer cell lines [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B27.

1634P - FGFR pathway genomic aberrations and response to FGFRs inhibitors

1634P - FGFR pathway genomic aberrations and response to FGFRs inhibitors

Conserved signaling mechanisms in Drosophila heart development.

Signal transduction through multiple distinct pathways regulates and orchestrates the numerous biological processes comprising heart development. This review outlines the roles of the FGFR, EGFR, Wnt, BMP, Notch, Hedgehog, Slit/Robo, and other signaling pathways during four sequential phases of Drosophila cardiogenesis-mesoderm migration, cardiac mesoderm establishment, differentiation of the cardiac mesoderm into distinct cardiac cell types, and morphogenesis of the heart and its lumen based on the proper positioning and cell shape changes of these differentiated cardiac cells-and illustrates how these same cardiogenic roles are conserved in vertebrates. Mechanisms bringing about the regulation and combinatorial integration of these diverse signaling pathways in Drosophila are also described. This synopsis of our present state of knowledge of conserved signaling pathways in Drosophila cardiogenesis and the means by which it was acquired should facilitate our understanding of and investigations into related processes in vertebrates. Developmental Dynamics 246:641-656, 2017. © 2017 Wiley Periodicals, Inc.

Abstract 3198: Using a genome-wide CRISPR-Cas9 knockout library to identify therapeutic combinations in oral cancer

Abstract Oral squamous cell carcinoma (OSCC) has remained a disease with poor survival for decades with few novel treatment options. We anticipate great potential for personalized targeted therapies, facilitated by recent characterizations of the mutational landscape of OSCCs. Combinations of targeted therapies may have greater efficacy through inhibiting compensatory pathways. For example, targeting EGFR has had limited success. To identify synergistic combinations with EGFR-targeted therapy, we introduced Genome-scale CRISPR-Cas9 Knock-Out (GeCKO) libraries into OSCC cell lines. CRISPR-Cas9 generates individual genetic knockouts through targeted gene editing. We used a pool of CRISPRs targeting over 18,000 genes to perform genome-scale screening for drivers of sensitivity to EGFR-targeted therapy. Upon selection of the OSCC GeCKO pool, we identified gene knockouts in the FGFR pathway that increased sensitivity to the EGFR inhibitor gefitinib. Using resazurin viability assays we tested combinations of EGFR and FGFR inhibitors in 14 OSCC cell lines. Six/14 (43%) of the cell lines were responsive to the combination, indicating that the FGFR pathway is an alternate mechanism of resistance to EGFR-targeted therapy in some tumors. In complement, we again used CRISPR-Cas9 to generate a syngeneic EGFR knockout (KO) OSCC cell line. The parent OSCC cell line is responsive to the EGFR and FGFR dual inhibition, and the EGFR KO derivative retains sensitivity to FGFR-targeted monotherapies. We expect that this EGFR KO cell line will be a useful tool in further evaluating the compensatory mechanism of the FGFR pathway. OSCC remains a common and frequently lethal cancer with great potential for the development of personalized targeted therapies. Here, we describe the use of genome-wide CRISPR-Cas9 library to discover the synergistic combination of EGFR and FGFR inhibition. Further investigation suggests the FGFR pathway is a common compensatory mechanism to EGFR inhibition. We hope to use this approach to identify additional compensatory mechanisms of resistance to targeted therapies with the eventual goal of translating these findings to clinic. Citation Format: Megan Ludwig, Andrew Birkeland, Sai Nimmagadda, Sue Foltin, Aditi Kulkarni, Hui Jiang, Thomas Carey, Chad Brenner. Using a genome-wide CRISPR-Cas9 knockout library to identify therapeutic combinations in oral cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3198. doi:10.1158/1538-7445.AM2017-3198

Abstract 1005: Interaction between the estrogen receptor and fibroblast growth factor receptor pathways in non-small cell lung cancer

Abstract The estrogen receptor (ER) promotes cell proliferation in non-small cell lung cancer (NSCLC). Since fibroblast growth factors (FGFs) are known regulators of stem cell markers in ER positive breast cancer, we investigated whether a link between the estrogen pathway, FGFs, and stem cell markers could be demonstrated in NSCLC animal models, human cells lines and tumor tissue. In the lungs of female FVB/N mice exposed to the tobacco carcinogen 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, NNK, treatment with the anti-estrogen fulvestrant and/or the aromatase inhibitor anastrozole blocked secretion of both FGF2 and FGF9, and reduced expression of the stem cell markers SOX2 and nanog. These effects were observed in the normal airway epilthelium as well as in both lung preneoplasias and lung adenomas. To investigate the effects of β-estradiol (E2), male mice were administered E2 in the drinking water during exposure to NNK for 4 weeks. In this mouse model, the incidence of carcinogen-induced lung preneoplasias increased 1.8-fold (p<0.05) with E2 exposure accompanied by increased expression of FGF2, FGF9, SOX2, and nanog in airway preneoplasias demonstrating a relationship between activation of the FGF pathway in the lungs, expansion of cells with a stem cell phenotype, and promotion of pre-cancerous changes. In FGFR1 normal copy number NSCLC cell lines that express multiple FGFR family members and secrete several FGFs, E2 treatment caused a significant increase (up to 4-fold; p<0.05) in release of FGF2, an effect that was completely blocked by fulvestrant. Treatment with fulvestrant also resulted in a 30% reduction in phosphorylation of fibroblast growth factor receptor substrate 2, the FGFR docking protein. Upon co-inhibition of ER and FGFRs using fulvestrant and the pan-FGFR inhibitor AZD4547, significantly enhanced (p<0.01) anti-proliferative effects were observed in FGFR1 normal copy number NSCLC cells that show low sensitivity to FGFR inhibitors as single agents. In NSCLC xenografts, the combination of AZD4547 (daily 12.5 mg/kg) and fulvestrant (30 mg/kg twice weekly) resulted in a significantly greater inhibition (67-85% decrease; p<0.05) of tumor growth and decreased expression of Ki67 and stem cell markers compared to each single agent treatment. Furthermore, tumor histology appeared more differentiated and up to a 25% decrease in malignant cellularity was observed with combination treatment compared to each single agent treatment. In NSCLC patient tumors, high ERB expression correlated with high FGFR1 expression (p<0.001). Taken together, these results suggest that interaction between the ER and FGFR pathways in NSCLC promotes a stem-like state. Combining an FGFR inhibitor with an ER pathway inhibitor could be exploited to increase the efficacy of FGFR inhibitors for NSCLC patients who lack FGFR genetic alterations. Supported by P50 CA090440 and the V-Foundation. Citation Format: Laura P. Stabile, Natalie J. Rothenberger, Mariya Farooqui, Sanja Dacic, Jill M. Siegfried. Interaction between the estrogen receptor and fibroblast growth factor receptor pathways in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1005. doi:10.1158/1538-7445.AM2017-1005