FGFR1 Activation Research Articles

Resistance to MET/VEGFR2 Inhibition by Cabozantinib Is Mediated by YAP/TBX5-Dependent Induction of FGFR1 in Castration-Resistant Prostate Cancer

The overall goal of this study was to elucidate the role of FGFR1 induction in acquired resistance to MET and VEGFR2 inhibition by cabozantinib in prostate cancer (PCa) and leverage this understanding to improve therapy outcomes. The response to cabozantinib was examined in mice bearing patient-derived xenografts in which FGFR1 was overexpressed. Using a variety of cell models that reflect different PCa disease states, the mechanism underpinning FGFR1 signaling activation by cabozantinib was investigated. We performed parallel investigations in specimens from cabozantinib-treated patients to confirm our in vitro and in vivo data. FGFR1 overexpression was sufficient to confer resistance to cabozantinib. Our results demonstrate transcriptional activation of FGF/FGFR1 expression in cabozantinib-resistant models. Further analysis of molecular pathways identified a YAP/TBX5-driven mechanism of FGFR1 and FGF overexpression induced by MET inhibition. Importantly, knockdown of YAP and TBX5 led to decreased FGFR1 protein expression and decreased mRNA levels of FGFR1, FGF1, and FGF2. This association was confirmed in a cohort of hormone-naïve patients with PCa receiving androgen deprivation therapy and cabozantinib, further validating our findings. These findings reveal that the molecular basis of resistance to MET inhibition in PCa is FGFR1 activation through a YAP/TBX5-dependent mechanism. YAP and its downstream target TBX5 represent a crucial mediator in acquired resistance to MET inhibitors. Thus, our studies provide insight into the mechanism of acquired resistance and will guide future development of clinical trials with MET inhibitors.

Compound 15c, a Novel Dual Inhibitor of EGFRL858R/T790M and FGFR1, Efficiently Overcomes Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor Resistance of Non-Small-Cell Lung Cancers.

In the past decades, epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) had been proved as an effective treatment strategy for the patients with EGFR-mutated non-small-cell lung cancer (NSCLC). However, the tolerance for the EGFR-TKI always occurred after continuous administration for a period of time and limiting the application of these drugs. Activation of FGFR1 signaling pathway was one of the important escape mechanisms for EGFR-TKI resistant in NSCLC. Here, a novel dual inhibitor of EGFRL858R/T790M and FGFR1, compound15c, was found and can efficiently overcame the EGFR-TKI resistance via its simultaneous inhibition of their kinase activities. Comparison with EGFRL858R/T790M and FGFR1 inhibitor treatment alone or combined revealed that the inhibition of EGFRL858R/T790M and FGFR1 activity by 15c was responsible for surmounting the intrinsic EGFR-TKI resistance in EGFRL858R/T790M-mutated H1975 cells and the acquired resistance in Afatinib-tolerant PC9 cells (AFA-PC9). Flow Cytometry and Caspase3 activity analysis assay showed that 15c induced significant the early apoptosis of H1975 cells. Xenograft tumor formation in BALB/c mice induced by a H1975 cells was suppressed by 15c treatment, with no changes in animal body weight. Generally, 15c may act as a new-generation EGFR-TKI for the therapy of NSCLC patients suffering a resistance to current TKI.

Identification and validation of poor prognosis immunoevasive subtype of muscle-invasive bladder cancer with tumor-infiltrating podoplanin+ cell abundance

ABSTRACT The choice of chemo- or immuno-therapy for muscle-invasive bladder cancer (MIBC) patients remains contentious. Podoplanin is newly identified as an immune checkpoint which intrigues us to explore the clinical significance and immunoregulatory role of tumor-infiltrating podoplanin+ cells (PDPN+ cells) in MIBC. A retrospective analysis of 259 MIBC patients from Zhongshan Hospital (n = 141) and Shanghai Cancer Center (n = 118) was conducted. A total of 406 MIBC patients from TCGA database were enrolled to investigate the relationship between PDPN and molecular characterization. We found that tumor-infiltrating PDPN+ cell abundance indicated an inferior overall survival and recurrence-free survival. pT2 MIBC patients with PDPN+ cell low infiltration could benefit more from adjuvant chemotherapy (ACT). Increased PDPN+ cell infiltration was associated with diminished GZMB and TNF-α expression while correlated with expanded PD-1, PD-L1, LAG-3 and TIM-3 expression and tumor-promoting regulatory T cell and M2 macrophage infiltration. Tumors with high PDPN mRNA expression mainly presented luminal-infiltrated and basal-squamous subtypes (2017 TCGA classification) or stroma-rich and Ba/Sq subtypes (consensus classification). Elevated PDPN mRNA expression was associated with less FGFR3 activation signature and more T-cell-inflamed signature and EGFR activation signature. In conclusion, tumor-infiltrating PDPN+ cells could be applied as an independent prognosticator for clinical outcome and a predictive biomarker for suboptimal ACT responsiveness, which was also associated with immunosuppressive contexture infiltration. Intratumoral PDPN expression had a correlation with MIBC molecular classification and therapy-related signatures. The novel immune checkpoint PDPN should be considered as a possible immunotherapeutic target for MIBC.

Polarizing receptor activation dissociates fibroblast growth factor 2 mediated inhibition of myelination from its neuroprotective potential

Fibroblast growth factor (FGF) signaling contributes to failure of remyelination in multiple sclerosis, but targeting this therapeutically is complicated by its functional pleiotropy. We now identify FGF2 as a factor up-regulated by astrocytes in active inflammatory lesions that disrupts myelination via FGF receptor 2 (FGFR2) mediated activation of Wingless (Wnt) signaling; pharmacological inhibition of Wnt being sufficient to abrogate inhibition of myelination by FGF2 in tissue culture. Using a novel FGFR1-selective agonist (F2 V2) generated by deleting the N-terminal 26 amino acids of FGF2 we demonstrate polarizing signal transduction to favor FGFR1 abrogates FGF mediated inhibition of myelination but retains its ability to induce expression of pro-myelinating and immunomodulatory factors that include Cd93, Lif, Il11, Hbegf, Cxcl1 and Timp1. Our data provide new insights into the mechanistic basis of remyelination failure in MS and identify selective activation of FGFR1 as a novel strategy to induce a neuroprotective signaling environment in multiple sclerosis and other neurological diseases.

Abstract C052: FGF trapping impairs multiple myeloma growth through c-Myc degradation-induced mitochondrial oxidative stress

Abstract Multiple myeloma (MM) remains incurable because of chemotherapeutic resistance. Fibroblast growth factors (FGF) play a pivotal role in MM by acting as an autocrine/paracrine mitogen on plasma cells, bone marrow-derived endothelial cells and fibroblasts. Also, a recurrent chromosomal translocation t(4;14) is associated with FGFR3 upregulation in MM patients. Thus, agents able to hamper FGF signaling may represent a novel approach for MM treatment. Recently we have identified the PTX3-derived small molecule NSC12 as the first orally active antitumor pan-FGF trap. Here, the role of FGF/FGFR system in MM cell survival and the therapeutic potential of NSC12 were investigated. In vitro effects of NSC12 were tested on four human MM cell lines harboring (KMS-11 and OPM-2 cells) or not (U-266 and RPMI8226 cells) the t(4:14) translocation and on patient-derived MM cells (N = 26). In vivo effects of FGF blockade were investigated using subcutaneous (KMS-11 and RPMI8226 cells) and systemic (MM-1S cells) xenografts grown in NOD/SCID mice. In addition, gene expression profiling (GEP) and gene set enrichment analyses (GSEA) were performed in NSC12-treated MM cells. NSC12 blocked the proliferation of all human MM cell lines tested, causing inhibition of FGFR signaling, downregulation of the anti-apoptotic protein mcl-1 and caspase 3 activation. Cytofluorimetric analysis revealed a significant increase of apoptotic cells as early as 6 hrs after NSC12 treatment, with 100% of cell death after 24 hrs even when MM cells were co-cultured with patient-derived bone marrow stromal cells. In vivo, NSC12 blocked FGFR activation and reduced the growth of subcutaneous xenografts and MM cell dissemination in the BM. GEP and GSEA analyses of NSC12-treated cells revealed the upregulation of oxidative stress-induced genes and the downmodulation of c-Myc targets. Accordingly, NSC12 caused the rapid proteasomal degradation of c-Myc paralleled by GSH depletion, mitochondrial ROS production and depolarization, DNA damage and finally apoptosis in KMS-11 cells but not in KMS-11 cells expressing the undegradable mutated (T58A) form of c-Myc. Interestingly, these findings were confirmed on Bortezomib-resistant MM cells as well as on bone marrow-derived primary MM cells from newly diagnosed and relapsed/refractory patients, including plasma cells bearing the t(4;14) translocation obtained from high-risk MM patients. Our data reveal that FGF blockade triggers MM mitochondrial oxidative stress, DNA damage and apoptotic cell death via the proteasomal degradation of the c-Myc oncoprotein. Altogether, our findings dissect for the first time the mechanism by which the FGF/FGFR system plays a non-redundant role in MM cell survival and disease progression, and indicate that FGF targeting may represent a therapeutic approach for MM patients with poor prognosis and advanced disease stage. Citation Format: Roberto Ronca, Gaia Cristina Ghedini, Federica Maccarinelli, Antonio Sacco, Silvia Laura Locatelli, Eleonora Foglio, Sara Taranto, Elisabetta Grillo, Sara Matarazzo, Riccardo Castelli, Vanessa Desantis, Nadia Cattane, Annamaria Cattaneo, Marco Mor, Carmelo Carlo-Stella, Aldo Maria Roccaro, Marco Presta, Arianna Giacomini. FGF trapping impairs multiple myeloma growth through c-Myc degradation-induced mitochondrial oxidative stress [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C052. doi:10.1158/1535-7163.TARG-19-C052

Abstract A118: Identification of resistance mechanisms to FGFR4 targeted therapy in hepatocellular carcinoma

Abstract When on-target resistance mutations occur in response to potent and selective therapeutics, the target is often considered a validated driver of disease. Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide whose drivers remain unvalidated because approved therapies utilize multi-kinase inhibitors or immune modulation. In this study, we validate for the first time a driver of disease in the FGF19 expressing subset of HCC through the identification of Fisogatinib resistance mutations in FGFR4. Fisogatinib (formerly known as BLU-554), an investigational agent, was developed to specifically inhibit the tyrosine kinase activity of FGFR4. Approximately one third of HCC patients harbor aberrant expression of fibroblast growth factor 19 (FGF19), a ligand hypothesized to drive paracrine FGFR4 activation and enhance mitogenic signaling in HCC cells. The identification of FGF19 amplifications in a subset of HCC patients through genomic profiling is further suggestive of the involvement of the FGF19/FGFR4 signaling axis in oncogenesis. The first-in-human, phase I study of Fisogatinib in patients with advanced HCC (NCT02508467) suggests that expression of FGF19 as assessed by immunohistochemistry is responsible for oncogenesis and predictive of response to treatment. We identified FGFR4 gatekeeper (V550) and hinge-1 (C552) mutations upon disease progression in two patients treated with Fisogatinib. These resistance mutations had been predicted by our structure-based studies and were also identified through in vitro and in vivo Fisogatinib resistance screens. We confirm by liquid chromatography tandem mass-spectrometry that Fisogatinib binding to FGFR4 is prevented when either the gatekeeper or the hinge-1 mutation is present and demonstrate the capability of these mutations to mediate resistance to Fisogatinib through genetic engineering. Using a gatekeeper-agnostic pan-FGFR inhibitor we show, in preclinical models, continued FGF19/FGFR4 pathway dependence. These results validate FGF19/FGFR4 as an oncogenic driver and inform the profile of potential next-generation inhibitors designed to facilitate improved patient outcomes. Citation Format: Megan A Hatlen, Oleg Schmidt-Kittler, Cori-Ann Sherwin, Emily Rozsahegyi, Nooreen Rubin, Michael Sheets, Joseph L Kim, Chandra Miduturu, Neil Bifulco, Natasja Brooijmans, Hongliang Shi, Timothy Guzi, Andy Boral, Christoph Lengauer, Marion Dorsch, Richard D Kim, Yoon-Koo Kang, Beni B Wolf, Klaus P Hoeflich. Identification of resistance mechanisms to FGFR4 targeted therapy in hepatocellular carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A118. doi:10.1158/1535-7163.TARG-19-A118

Sprouty4 correlates with favorable prognosis in perihilar cholangiocarcinoma by blocking the FGFR-ERK signaling pathway and arresting the cell cycle.

BackgroundPerihilar cholangiocarcinoma (PHCC) is the most common subtype of cholangiocarcinoma(CCA). We previously investigated the expression pattern of Sprouty(SPRY) in intrahepatic cholangiocarcinoma(ICC), but the expression and clinical significance of SPRY family members in PHCC are still unknown.MethodsThe expression of SPRY family members(SPRY1-4) was detected in different subtypes of CCA and corresponding adjacent tissues. SPRY4 expression in 142 cases of PHCC was detected by immunohistochemistry, and its clinical significance was evaluated using univariate and multivariate analyses. The functions of SPRY4 in the FGFR-induced proliferation and migration of PHCC cells were investigated through in vitro and in vivo experiments. We further investigated the effects and mechanisms of SPRY4 on FGFR-induced ERK phosphorylation and cell cycle distribution in the presence of FGFR and ERK inhibitors.FindingsSPRY4 was the only SPRY family member associated with PHCC prognosis, and it was identified as an independent factor predicting favorable prognosis. In PHCC, SPRY4 expression was extensively associated with FGFR2, and its expression can be induced by ectopic FGFR2 activation. Through in vitro and in vivo experiments, we demonstrated that SPRY4 suppressed FGFR-induced proliferation and migration by inhibiting ERK phosphorylation. Moreover, SPRY4 knockdown was shown to decrease the percentage of cells in the G1 phase and promote the percentage of cells in the S and G2/M phases by increasing cyclin D1 expression, which also required FGFR-induced ERK phosphorylation.InterpretationHigh expression of SPRY4 was an independent biomarker of favorable prognosis in PHCC. SPRY4 expression can be induced by ectopic FGFR2 activation in PHCC. SPRY4 arrested the cell cycle at G1 phase and suppressed FGFR-induced proliferation and migration by inhibiting ERK phosphorylation, indicating that SPRY4 may be a potential therapeutic target in PHCC.

Long non-coding RNAs identify a subset of luminal muscle-invasive bladder cancer patients with favorable prognosis

BackgroundMuscle-invasive bladder cancer (MIBC) is a heterogeneous disease, and gene expression profiling has identified several molecular subtypes with distinct biological and clinicopathological characteristics. While MIBC subtyping has primarily been based on messenger RNA (mRNA), long non-coding RNAs (lncRNAs) may provide additional resolution.MethodsLncRNA expression was quantified from microarray data of a MIBC cohort treated with neoadjuvant chemotherapy (NAC) and radical cystectomy (RC) (n = 223). Unsupervised consensus clustering of highly variant lncRNAs identified a four-cluster solution, which was characterized using a panel of MIBC biomarkers, regulon activity profiles, gene signatures, and survival analysis. The four-cluster solution was confirmed in The Cancer Genome Atlas (TCGA) cohort (n = 405). A single-sample genomic classifier (GC) was trained using ridge-penalized logistic regression and validated in two independent cohorts (n = 255 and n = 94).ResultsNAC and TCGA cohorts both contained an lncRNA cluster (LC3) with favorable prognosis that was enriched with tumors of the luminal-papillary (LP) subtype. In both cohorts, patients with LP tumors in LC3 (LPL-C3) were younger and had organ-confined, node-negative disease. The LPL-C3 tumors had enhanced FGFR3, SHH, and wild-type p53 pathway activity. In the TCGA cohort, LPL-C3 tumors were enriched for FGFR3 mutations and depleted for TP53 and RB1 mutations. A GC trained to identify these LPL-C3 patients showed robust performance in two validation cohorts.ConclusionsUsing lncRNA expression profiles, we identified a biologically distinct subgroup of luminal-papillary MIBC with a favorable prognosis. These data suggest that lncRNAs provide additional information for higher-resolution subtyping, potentially improving precision patient management.

Critical individual roles of the BCR and FGFR1 kinase domains in BCR-FGFR1-driven stem cell leukemia/lymphoma syndrome.

Constitutive activation of FGFR1, as a result of diverse chromosome translocations, is the hallmark of stem cell leukemia/lymphoma syndrome. The BCR-FGFR1 variant is unique in that the BCR component contributes a serine-threonine kinase (STK) to the N-terminal end of the chimeric FGFR1 kinase. We have deleted the STK domain and mutated the critical Y177 residue and demonstrate that the transforming activity of these mutated genes is reduced compared to the BCR-FGFR1 parental kinase. In addition, we demonstrate that deletion of the FGFR1 tyrosine kinase domain abrogates transforming ability, which is not compensated for by BCR STK activity. Unbiased screening for proteins that are inactivated as a result of loss of the BCR STK identified activated S6 kinase and SHP2 kinase. Genetic and pharmacological inhibition of SHP2 function in SCLL cells expressing BCR-FGFR1 in vitro leads to reduced viability and increased apoptosis. In vivo treatment of SCLL in mice with SHP099 leads to suppression of leukemogenesis, supporting an important role for SHP2 in FGFR1-driven leukemogenesis. In combination with the BGJ398 FGFR1 inhibitor, cell viability in vitro is further suppressed and acts synergistically with SHP099 in vivo suggesting a potential combined targeted therapy option in this subtype of SCLL disease.

905PD - Multiple-cohort analysis investigating FGFR3 alteration as a predictor of non-response to neoadjuvant pembrolizumab (pembro) in muscle-invasive bladder cancer (MIBC)

BackgroundIn PURE01 study (NCT02736266), neoadjuvant pembro resulted in 42% pT0 in patients (pts) with MIBC. pT0 pts had features suggesting pre-existing immunity or higher tumor mutational burden (TMB) may promote response. In this study, we investigated potential mechanisms for resistance to pembro, including FGFR3 genomic alterations (GA). MethodsPts enrolled in the PURE-01, which is still recruiting pts in its amended design, had predominant urothelial carcinoma histology and stage cT≤4N0 MIBC. Biomarker analyses in the expanded cohort of 96 pts included PD-L1 combined positive score and comprehensive genomic profiling (FoundationCDx assay). In addition, TCGA MIBC (n=405) and a prospective commercial cohort (PCC) of 415 MIBC pts from the clinical use of the Decipher Bladder TURB test from the GRID registry (NCT02609269) were analyzed. A single-sample genomic classifier (GC) was trained to identify FGFR3-active tumors (FGFR3+). ResultsIn PURE01 cohort, despite a linear association of TMB with ypT0 in multivariable models (p=0.017), there was no association between FGFR3 GA and pathological response nor with PD-L1 expression. FGFR3 GA were found in 17.6% ypT0 vs 20.7% in ypT3-4 disease (corrected p=0.82). Applying the GC to the TCGA MIBC cohort, we found 45% of FGFR3+ cases had FGFR3 mutations vs 9% for the rest of the cohort. Applying the GC to the PCC, we found that the FGFR3+ tumors were Luminal (n=14), Luminal Infiltrated (n=10) or Luminal Papillary (n=30). FGFR3+ pts showed significantly lower PD-L1 (-0.03 vs. 0.109, p<0.001) and PD-L2 (0.184 vs. 0.389, p<0.001), but consistent PD-1 (0.175 vs. 0.163, p=0.48) gene expression. FGFR3+ pts had lower immune and stromal infiltration as measured by the immune190 (0.227 vs. 0.348, p<0.001) and Estimate (100.14 vs. 1099.2, p<0.001) gene expression signatures. ConclusionsIn our combined cohorts, FGFR3 GA did not correlate with response to pembro. FGFR3 GA are enriched in FGFR3+ tumors, which in turn tend to have lower immune activity, PD-L1 and PD-L2, suggesting FGFR3 activity may provide a potential tool for further discriminating the mechanisms underlying response and resistance to neoadjuvant pembro in MIBC. Clinical trial identificationNCT02736266 (PURE01) NCT02609269 (GRID Registry). Legal entity responsible for the studyThe authors. FundingDecipher Bioscience. DisclosureA. Necchi: Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Merck; Honoraria (institution), Advisory / Consultancy: Roche; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: BMS; Advisory / Consultancy, Research grant / Funding (institution): Incyte; Advisory / Consultancy: Clovis; Advisory / Consultancy: Janssen. R. Madison: Full / Part-time employment: Foundation Medicine. J. Chung: Full / Part-time employment: Foundation Medicine. Y. Liu: Full / Part-time employment: Decipher Bioscience. J. Chung: Full / Part-time employment: Foundation Medicine. J.S. Ross: Full / Part-time employment: Foundation Medicine. S.M. Ali: Full / Part-time employment: Foundation Medicine. E. Davicioni: Full / Part-time employment: Decipher Bioscience. E.A. Gibb: Full / Part-time employment: Decipher Bioscience. All other authors have declared no conflicts of interest.

FGFR4 does not contribute to progression of chronic kidney disease

In chronic kidney disease (CKD), elevated serum levels of the phosphate regulating hormone fibroblast growth factor (FGF) 23 have emerged as powerful risk factors for cardiovascular disease and death. Mechanistically, FGF23 can bind and activate fibroblast growth factor receptor (FGFR) 4 independently of α-klotho, the canonical co-receptor for FGF23 in the kidney, which stimulates left ventricular hypertrophy and hepatic production of inflammatory cytokines. FGF23 has also been shown to independently predict progression of renal disease, however, whether FGF23 and FGFR4 also contribute to CKD remains unknown. Here, we generated a mouse model with dual deletions of FGFR4 and α-klotho, and we induced CKD in mice with either global deletion or constitutive activation of FGFR4. We demonstrate that FGF23 is not capable of inducing phosphaturia via FGFR4 and that FGFR4 does not promote or mitigate renal injury in animal models of CKD. Taken together our results suggest FGFR4 inhibition as a safe alternative strategy to target cardiovascular disease and chronic inflammation in patients with CKD without interrupting the necessary phosphaturic effects of FGF23.

Integrated Clinico-Molecular Characterization Identifies RAS Activation and CDKN2A Deletion As Independent Adverse Prognostic Factors in Cancer of Unknown Primary

Background: Cancer of unknown primary (CUP) denotes a malignancy with histologically confirmed metastatic spread while the primary tumor remains elusive. Here, we address prognostic and therapeutic implications of mutations and copy number variations (CNVs) detected in tumor tissue in the context of a comprehensive clinical risk assessment. Methods: Targeted panel sequencing was performed in 252 CUP patients. 71.8% of patients had unfavorable CUP according to ESMO guidelines. 74.7% were adeno- and 13.7% squamous cell carcinomas. DNA was extracted from micro-dissected formalin-fixed, paraffin-embedded tissues. For library preparation, mostly multiplex PCR-based Ion Torrent AmpliSeqTM technology with Oncomine comprehensive assays was used. Findings: Most frequent genetic alterations were mutations/deletions of TP53 (49.6%), CDKN2A (19.0%) and NOTCH1 (14.1%) as well as oncogenic activation of KRAS (23.4%), FGFR4 (14.9%) and PIK3CA (10.7%). KRAS activation was predominantly found in adenocarcinomas (P=0.01), PIK3CA activation in squamous cell carcinomas (P=0.03). Male sex, high ECOG score, unfavorable CUP, higher number of involved organs and RAS activation predicted decreased event-free and overall survival in multivariate analysis. Deletions of CDKN2A were prognostically adverse regarding overall survival. TP53 mutations did not significantly influence prognosis in the overall cohort, but worsened prognosis in otherwise favorable CUP subtypes. Although not standard in CUP, for 17/198 (8.6%) patients molecularly targeted treatment was recommended and 10 patients (5.1%) were treated accordingly. Interpretation: Besides the identification of drug targets, panel sequencing in CUP is prognostically relevant, with RAS activation and CDKN2A deletion emerging as novel independent risk factors in a comprehensive assessment with clinico-pathological data. Funding: None. Declaration of Interest: TB and AK work as study oncologists for the CUPISCO trial, which is sponsored by Roche, and have received reimbursement for study‐related travels as well as remuneration for their work as study oncologists for the benefit of their employer. VE: advisory board and lecture fees from AstraZeneca and ThermoFisher. ALV: lecture fees from AstraZeneca. PS: advisory board honoraria from Pfizer, Roche, Novartis and AstraZeneca as well as speaker's honoraria and research funding from Roche, AstraZeneca and Novartis. WW: advisory board honoraria from MSD, BMS, Roche, AZ, Novartis, Celgene, Merck, Pfizer; speaker's honoraria from MSD, BMS, Roche, AZ, Boehringer, Lilly, Amgen, Takeda, Novartis; research funding (to institution): Roche, MSD, BMS. AS: advisory board honoraria from Bayer, Novartis, BMS, AstraZeneca, ThermoFisher, Illumina; speaker's honoraria from Takeda, Roche, BMS, Illumina, AstraZeneca, Novartis, ThermoFisher, Bayer, MSD and research funding from Chugai and BMS. All remaining authors have declared no conflicts of interest. Ethical Approval: Ethics board approval from the University of Heidelberg was obtained and patients signed informed consent.

Abstract 2868: CD99 inhibition by clofarabine induces Ewing sarcoma cytotoxicity through activation of FGFR1 and downstream kinase pathways

Abstract Ewing sarcoma (ES) cells express high levels of the cell surface protein CD99, which is used for diagnosis of ES. More importantly, the inhibition of CD99 activity results in reduced growth of ES cells in vitro and in vivo. In an earlier publication, we have identified clofarabine and cladribine as selective inhibitors of CD99, which inhibited ES growth both in vitro and in vivo. Clofarabine and cladribine directly bound to CD99, inhibited its molecular interactions, and regulated CD99 specific intracellular signaling pathways. In order to gain further insight into how clofarabine may regulate cellular functions through inhibiting CD99, we utilized a phospho-kinase array to identify changes in phosphorylation levels of 43 proteins in response to clofarabine or CD99 antibody treatment. We identified ERK1/2-MSK1/2-CREB signaling axis as one of the signaling pathways downstream of CD99. These findings were validated in four different ES cell lines and in xenograft lysates that were harvested from clofarabine-treated mice compared to control mice. The phosphorylation events induced by clofarabine treatment was significantly diminished when CD99 was knocked down either by siRNA-mediated knockdown or CRISPR/Cas9-mediated genomic disruption. Time-course experiments on ES cells showed that clofarabine triggers a very rapid signaling cascade through CD99. Cytarabine, a structurally similar pyrimidine analog and an inhibitor of the EWS-FLI1 transcriptional activity that has been failed in clinical trials on ES patients, did not activate ERK1/2, MSK1/2 or CREB phosphorylation in ES cells. In order to test whether the observed changes in phosphorylation levels are required for cell death, we screened a kinase inhibitor small molecule library for their ability to rescue clofarabine-induced death of ES cells. We identified TG100-115 (PI3K inhibitor) and rebastinib (c-abl inhibitor) as the most potent kinase inhibitors that showed significant reversal of clofarabine-induced ES cell death, suggesting that clofarabine-induced death of ES cells requires specific phosphorylation cascades. We then discovered that CD99 can be co-immunoprecipitated with FGFR1 and clofarabine treatment of ES cells results in phosphorylation of FGFR1. In conclusion, we discovered a novel mechanism of action for clofarabine and cladribine in that their selective cytotoxic effect on ES is through inhibiting CD99 on the cell surface, and it is not dependent on their ability to inhibit DNA synthesis. CD99 inhibition resulted in increased phosphorylation of FGFR and two downstream signaling pathways (PI3K/Akt and MEK1/ERK1). Inhibition of PI3K pathway rescues clofarabine-induced ES cell cytotoxicity. These findings provide additional mechanistic support for repurposing clofarabine and cladribine for ES indication. Note: This abstract was not presented at the meeting. Citation Format: Haydar Celik, Levent Dusunceli, Anna Molotkova, David V. Allegakoen, Erin J. Conn, Jeff R. Petro, Jeffrey A. Torestky, Aykut Uren. CD99 inhibition by clofarabine induces Ewing sarcoma cytotoxicity through activation of FGFR1 and downstream kinase pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2868.

Abstract 4339: Critical role of Dnmt3b catalytic activity in prevention of oncogene-induced leukemias and lymphomas

Abstract Cytosine methylation is involved in a variety of biological processes, including development, X-chromosome inactivation, imprinting, and suppressing unwanted transcription. DNA methylation in mammalian cells is catalyzed by several DNA methyltransferases, including DNMT1, DNMT3A and DNMT3B. In addition to DNA methylation catalysis, these enzymes interact with a number of repressive proteins including HDACs, thereby repressing transcription in methylation-independent manner. The extent to which methylation-dependent and independent activities play roles in biological processes remains poorly understood. Here we examined the role of methyltransferase (MT) activity of Dnmt3b in normal mouse embryogenesis and in prevention of hematologic malignancies. Loss of Dnmt3b in mice is embryonically lethal between 10.5-12.5 dpc. Dnmt3b was also identified as tumor suppressor in murine models of lymphoid and myeloid malignancies. To address whether Dnmt3b’s catalytic activity is important for mouse embryogenesis and prevention of hematologic malignancies, we generated mice bearing a conventional Dnmt3bCI allele using CRISPR/Cas9 system. This catalytically inactive allele of Dnmt3b (Dnmt3bCI) was generated by a double amino acid substitution in enzyme active center (P656V and C657D) abolishing its ability to perform transfer of methyl group onto cytosine. Surprisingly, Dnmt3bCI/CI mice were born at similar ratios as wild-type littermates and lived over 12 months suggesting that Dnmt3b is dispensable for both pre- and post-natal development. Importantly, MYC-induced T-cell lymphomagenesis was accelerated in the absence of Dnmt3b’s methyltransferase activity in MYC;Dnmt3bCI/CI mice. Global gene expression and methylation profiling revealed a number of deregulated events specific for MYC;Dnmt3bCI/CI, resulting in activation of Pak, Fgfr and Igf2 signaling and downregulation of tumor suppressors (e.g. Lrp12, Dusp6 and Marcks). The lack of Dnmt3b catalytic activity also accelerated a development of acute myeloid leukemia induced by MLL-AF9 overexpression in mice. Altogether, our data suggest that Dnmt3b’s MT activity is dispensable for mouse development but critical to prevent hematologic malignancies. Citation Format: Katarina Lopusna, Pawel Nowialis, Staci L. Haney, Ajay Abraham, Jana Opavska, Rene Opavsky. Critical role of Dnmt3b catalytic activity in prevention of oncogene-induced leukemias and lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4339.

Abstract 1736: Targeting Hsp90-Cdc37 complex overcomes drug resistance in glioma cells harboring FGFR3-TACC3 fusion gene

Abstract Glioblastoma is one of the most lethal malignancies with the poorest prognosis among tumors of the central nervous system. Fusion genes are common chromosomal aberrations in many cancers that may give rise to an in-frame fusion protein with oncogenic function. These fusion genes can be used as prognostic markers as well as drug targets in clinical practice. The FGFR3-TACC3 (F3T3) fusion gene was discovered in glioblastoma, with an occurrence rate of up to 8.3% and leading to uncontrolled proliferation and chromosomal instability. Our studies have shown that glioblastoma cells harboring the F3T3 are resistant to the frontline glioblastoma drug Temozolomide (TMZ). Using the Comet assay, we show that TMZ-mediated DNA damage is repaired more rapidly in cells harboring the F3T3 compared to control. Our studies on the mechanism of this drug resistance show that the F3T3 confers an aberrant activation of FGFR and ERK pathways when treated with TMZ. To further explore druggable target(s) to overcome resistance caused by F3T3, we immunoprecipitated (IP) the proteins that interact with F3T3 in U251 glioblastoma cells overexpressing the F3T3 or wildtype FGFR3, followed by LC-MS/MS analyses. Our quantitative proteomic analysis revealed interactions of the F3T3 fusion protein with Hsp90α and Cdc37 proteins. These interactions were further validated by reciprocal IPs followed by Western blotting. Activation of many kinases, such as FGFRs, depends on their interaction with the Hsp90 molecular chaperone system. Hsp90 recruitment is mediated by the co-chaperone adaptor protein Cdc37, which simultaneously binds to both the kinase and Hsp90. To test inhibition of F3T3 association with the Hsp90-Cdc37 complex, we treated U251 cells constitutively expressing F3T3 with the Hsp90 inhibitor Onalespib. We show that Onalespib treatment at 26 nM significantly suppresses the proliferation of F3T3 expressing U251 cells in comparison to micromolar levels of the pan-FGFR inhibitor BGJ398 and PD173074. Our results provide evidence that the F3T3 fusion gene contributes to drug resistance via multiple chemo-resistance pathways. Importantly, our findings establish that F3T3 is an Hsp90 client that shows strong addiction to the Hsp90-Cdc37 complex for cell growth and suggests a novel strategy for targeting F3T3 fusion gene in glioma. Citation Format: Tao Li, Farideh Mehraein-Ghomi, Sanjeev V. Namjoshi, Lynette M. Phillips, Elizabeth A. Ballard, Mary E. Forbes, ping-Chieh Chou, Xuejun Yang, Wei Zhang. Targeting Hsp90-Cdc37 complex overcomes drug resistance in glioma cells harboring FGFR3-TACC3 fusion gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1736.

Abstract LB-259: FGFR- and EGFR-driven SHP2 activation displays differential sensitivity to allosteric SHP2 inhibition

Abstract Introduction: The non-receptor protein tyrosine phosphatase SHP2 mediates RAS activation downstream of various receptor tyrosine kinases (RTKs). Cancer cell lines dependent on RTK signaling are generally sensitive to SHP2 genetic knockdown but differ widely in their sensitivity to allosteric SHP2 inhibitors such as SHP099. A recent report suggested that FGFR can drive resistance to MAPK inhibitors independently of SHP2. We sought to understand the molecular basis of differential sensitivity to allosteric SHP2 inhibitors when SHP2 is activated by different RTKs, particularly FGFR versus EGFR. Experimental procedures: We profiled a panel of cancer cell lines driven by various wild-type FGFR activation (receptor amplification or autocrine ligand mediated) for their sensitivity to SHP099 and characterized MAPK pathway inhibition at various time points and SHP099 doses, in comparison with EGFR-driven cell lines. We also compared the inhibitory effects of SHP099 on FGFR- and EGFR-mediated pathway feedback activation in response to MEK inhibitor treatment. Results: We find FGFR-driven cell lines are often more resistant to SHP099 compared to EGFR-driven cell lines. Those SHP099-resistant FGFR-driven cell lines were sensitive to SHP2 knockdown and SHP099 inhibited p-ERK in those cells at earlier time points. However, p-ERK quickly rebounded after SHP099 treatment, which was not observed with FGFR inhibitors and can be attenuated with high concentrations of SHP099. Consistently, FGFR-driven MAPK pathway feedback activation following treatment with MEK inhibitor was also more resistant to SHP099 compared to EGFR-driven feedback activation. Additional studies are being performed in isogenic cell lines for direct comparison. Conclusions: Our findings suggest that many FGFR-driven cell lines are dependent on SHP2 but are intrinsically resistant to allosteric SHP2 inhibition, compared to EGFR-driven cell lines. FGFR-driven cells tend to have a quick p-ERK rebound following initial pathway inhibition by SHP099, which is mediated by the feedback activation of FGFR. The feedback activated FGFR likely efficiently promotes the open conformation of SHP2 and causes resistance to allosteric SHP2 inhibitors. For certain FGFR-driven cancers, SHP2 inhibitor combinations such as with MAPK inhibitors might be needed for sustained efficacy. Citation Format: Huaixiang Hao, Hengyu Lu, Chen Liu, Matthew LaMarche, Jeffrey A. Engelman, Peter S. Hammerman, Giordano Caponigro. FGFR- and EGFR-driven SHP2 activation displays differential sensitivity to allosteric SHP2 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-259.

Abstract 2206: Anti-tumor activity of infigratinib, a potent and selective inhibitor of FGFR1, FGFR2 and FGFR3, in FGFR fusion-positive cholangiocarcinoma and other solid tumors

Abstract Abnormal expression and constitutive activation of receptor tyrosine kinases, such as ALK, ROS1 and TRK, as a result of gene rearrangements have been clinically validated as therapeutic targets for cancer. Recently, fusions involving the FGFR family, especially FGFR1, FGFR2 and FGFR3, have been identified in diverse solid tumors such as cholangiocarcinoma, glioblastoma, bladder, lung, breast, thyroid and prostate cancers. FGFR fusions are oncogenic drivers that ligand-independently activate the receptor kinases and their downstream signaling pathways, leading to uncontrolled cell proliferation and invasion. Infigratinib (BGJ398) is an oral and selective inhibitor of FGFR. At biochemical and cellular levels, infigratinib selectively inhibits the activity of FGFR1, FGFR2 and FGFR3 with low nM potency, while sparing FGFR4, VEGFR2 and other kinases. In FGFR fusion-positive xenograft models, including cell line-derived and patient-derived models representing diverse tumor histologies, infigratinib treatment leads to significant tumor growth inhibition and regression at clinically achievable exposure levels. The anti-tumor effect of infigratinib is accompanied by inhibition of downstream signaling pathways and induction of tumor apoptosis. Clinically, in an open-label phase II trial, infigratinib demonstrated a cORR of 39.3% in FGFR2 fusion-positive cholangiocarcinoma patients who had received one or fewer prior chemotherapy regimens. Additionally, RECIST responses were achieved in non-cholangiocarcinoma solid tumors tested positive for FGFR fusions. In conclusion, the available preclinical and clinical data demonstrate the potential of infigratinib as an effective treatment for patients with FGFR fusion-positive tumors, regardless of the fusion partners or the origin of tissue. On the basis of these data there is a clear rationale for performing tumor-agnostic clinical trials in molecularly defined cancers to maximally benefit patients with serious and life-threatening diseases. Citation Format: Gary Li, Melanie Krook, Sameek Roychowdhury, Francesca Avogadri, Yining Ye, Susan Moran. Anti-tumor activity of infigratinib, a potent and selective inhibitor of FGFR1, FGFR2 and FGFR3, in FGFR fusion-positive cholangiocarcinoma and other solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2206.

Fibroblast growth factor receptor promotes progression of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte-derived invasive and metastatic tumor of the skin. It is the second-most commonly diagnosed form of skin cancer striking 200 000 Americans annually. Further, in organ transplant patients, there is a 65- to 100-fold increased incidence of cSCC compared to the general population. Excision of cSCC of the head and neck results in significant facial disfigurement. Therefore, increased understanding of the mechanisms involved in the pathogeneses of cSCC could identify means to prevent, inhibit, and reverse this process. In our previous studies, inhibition of fibroblast growth factor receptor (FGFR) significantly decreased ultraviolet B-induced epidermal hyperplasia and hyperproliferation in SKH-1 mice, suggesting an important role for FGFR signaling in skin cancer development. However, the role of FGFR signaling in the progression of cSCC is not yet elucidated. Analysis of the expression of FGFR in cSCC cells and normal epidermal keratinocytes revealed protein overexpression and increased FGFR2 activation in cSCC cells compared to normal keratinocytes. Further, tumor cell-specific overexpression of FGFR2 was detected in human cSCCs, whereas the expression of FGFR2 was low in premalignant lesions and normal skin. Pretreatment with the pan-FGFR inhibitor; AZD4547 significantly decreased cSCC cell-cycle traverse, proliferation, migration, and motility. Interestingly, AZD4547 also significantly downregulated mammalian target of rapamycin complex 1 and AKT activation in cSCC cells, suggesting an important role of these signaling pathways in FGFR-mediated effects. To further bolster the in vitro studies, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice with SCC12A tumor xenografts treated with AZD4547 (15 mg/kg/bw, twice weekly oral gavage) exhibited significantly decreased tumor volume compared to the vehicle-only treatment group. The current studies provide mechanistic evidence for the role of FGFR and selectively FGFR2 in the early progression of cSCC and identifies FGFR as a putative therapeutic target in the treatment of skin cancer.

FGFR4 overexpression and hotspot mutations in metastatic ER+ breast cancer are enriched in the lobular subtype

Invasive lobular carcinoma (ILC) is an understudied subtype of breast cancer that requires novel therapies in the advanced setting. To study acquired resistance to endocrine therapy in ILC, we have recently performed RNA-Sequencing on long-term estrogen deprived cell lines and identified FGFR4 overexpression as a top druggable target. Here, we show that FGFR4 expression also increases dramatically in endocrine-treated distant metastases, with an average fold change of 4.8 relative to the paired primary breast tumor for ILC, and 2.4-fold for invasive ductal carcinoma (IDC). In addition, we now report that FGFR4 hotspot mutations are enriched in metastatic breast cancer, with an additional enrichment for ILC, suggesting a multimodal selection of FGFR4 activation. These data collectively support the notion that FGFR4 is an important mediator of endocrine resistance in ILC, warranting future mechanistic studies on downstream signaling of overexpressed wild-type and mutant FGFR4.

Identification of metabolic vulnerabilities of receptor tyrosine kinases-driven cancer

One of the biggest hurdles for the development of metabolism-targeted therapies is to identify the responsive tumor subsets. However, the metabolic vulnerabilities for most human cancers remain unclear. Establishing the link between metabolic signatures and the oncogenic alterations of receptor tyrosine kinases (RTK), the most well-defined cancer genotypes, may precisely direct metabolic intervention to a broad patient population. By integrating metabolomics and transcriptomics, we herein show that oncogenic RTK activation causes distinct metabolic preference. Specifically, EGFR activation branches glycolysis to the serine synthesis for nucleotide biosynthesis and redox homeostasis, whereas FGFR activation recycles lactate to fuel oxidative phosphorylation for energy generation. Genetic alterations of EGFR and FGFR stratify the responsive tumors to pharmacological inhibitors that target serine synthesis and lactate fluxes, respectively. Together, this study provides the molecular link between cancer genotypes and metabolic dependency, providing basis for patient stratification in metabolism-targeted therapies.