Receptor Tyrosine Kinase Mutations Research Articles

Predicting Response to Dasatinib Using a Computational Model and Its Validation: A Beat AML Project Study

Background: New prognostic factors have been recently identified in AML patient population that include frequent mutations of receptor tyrosine kinases (RTK) including KIT, PDGFR, FLT3, that are associated with higher risk of relapse. Thus, targeting RTKs could improve the therapeutic outcome in AML patients.Aim: To create a digital drug model for dasatinib and validate the predicted response in AML patient samples with ex vivo drug sensitivity testing.Methods: The Beat AML project (supported by the Leukemia & Lymphoma Society) collects clinical data and bone marrow specimens from AML patients. Bone marrow samples are analyzed by conventional cytogenetics, whole-exome sequencing, RNA-seq, and an ex vivo drug sensitivity assay. For 50 randomly chosen patients, every available genomic abnormality was inputted into a computational biology program (Cell Works Group Inc.) that uses PubMed and other online resources to generate patient-specific protein network maps of activated and inactivated pathways. Digital drug simulations with dasatinib were conducted by quantitatively measuring drug effect on a composite AML disease inhibition score (DIS) (i.e., cell proliferation, viability, and apoptosis). Drug response was determined based on a DIS threshold reduction of > 65%. Computational predictions of drug response were compared to dasatinib IC50 values from the Beat AML ex vivo testing.Results: 23/50 (46%) AML patients had somatic mutations in an RTK gene (KIT, PDGFR, FLT3 (ITD (n=15) & TKD (n=4)), while 27/50 (54%) were wild type (WT) for the RTK genes. Dasatinib showed ex vivo cytotoxicity in 9/50 (18%) AML patients and was predicted by CBM to remit AML in 9/50 AML patients with 4 true responders and 5 false positive. Ex vivo dasatinib responses were correctly matched to the CBM prediction in 40/50 (80%) of patients (Table1), with 10 mismatches due to lack of sufficient genomic information resulting in profile creation issues and absence of sensitive loops in the profile. Only 4/23 (17%) RTK-mutant patients and 5/27(19%) RTK-WT patients were sensitive to dasatinib ex vivo, indicating that presence of somatic RTK gene mutations may not be essential for leukemia regression in response to dasatinib. Co-occurrence of mutations in NRAS, KRAS and NF1 seemed to associate with resistance as seen in 10 of the 14 profiles harboring these mutations.Conclusion: Computational biology modeling can be used to simulate dasatinib drug response in AML with high accuracy to ex vivo chemosensitivity. DNA mutations in RTK genes may not be required for dasatinib response in AML. Co-occurrence of NRAS, KRAS and NF1gene mutations may be important co-factors in modulating response to dasatinib. [Display omitted] DisclosuresTyner:Leap Oncology: Equity Ownership; Syros: Research Funding; Seattle Genetics: Research Funding; Janssen: Research Funding; Incyte: Research Funding; Gilead: Research Funding; Genentech: Research Funding; AstraZeneca: Research Funding; Aptose: Research Funding; Takeda: Research Funding; Agios: Research Funding. Druker:Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; Millipore: Patents & Royalties; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; McGraw Hill: Patents & Royalties; Celgene: Consultancy; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Patient True Talk: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Beta Cat: Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy. Sahu:Cellworks Research India Private Limited: Employment. Vidva:Cellworks Research India Private Limited: Employment. Kapoor:Cellworks Research India Private Limited: Employment. Azam:Cellworks Research India Private Limited: Employment. Kumar:Cellworks Research India Private Limited: Employment. Chickdipatti:Cellworks Research India Private Limited: Employment. Raveendaran:Cellworks Research India Private Limited: Employment. Gopi:Cellworks Research India Private Limited: Employment. Abbasi:Cell Works Group Inc.: Employment. Vali:Cell Works Group Inc.: Employment. Cogle:Celgene: Other: Steering Committee Member of Connect MDS/AML Registry.

Activating Mutations in CSF1R and Additional Receptor Tyrosine Kinases in Sporadic and Familial Histiocytic Neoplasms

Genomic analyses of histiocytic neoplasms (including Langerhans Cell Histiocytosis (LCH) and the non-LCH Erdheim-Chester Disease (ECD)) have revolutionized our understanding of these disorders as clonal hematopoietic malignancies driven by MAPK signaling and led to FDA approval of vemurafenib for BRAFV600E-mutant ECD. Despite these advances, several questions about the pathogenesis of the histiocytoses remain unanswered. For example, the cell-of-origin of the histiocytoses is not definitively known. In addition, histiocytoses represent a spectrum of diseases, and genetic alterations across histiocytosis subtypes have not been comprehensively evaluated. Finally, although the histiocytoses most commonly occur as sporadic, non-hereditary disorders, familial clustering has been well documented and occurs most often in monozygotic twins. This has been taken to suggest a hereditary component of the disease, but germline genetic causes for histiocytoses are not known. Here we performed comprehensive genomic analyses of 218 patients with all subsets of histiocytoses, including monozygotic twins with histiocytosis. In so doing, we uncover a novel series of activating receptor tyrosine kinase (RTK) alterations in the histiocytoses, including the first example in any disease of recurrent, activating mutations in CSF1R, the RTK required for monocyte/macrophage development.We initially performed whole exome sequencing (WES) of skin lesions, blood, and fingernails from identical (monozygotic, dichorionic), one-year-old twins with systemic juvenile xanthogranuloma (JXG). This identified an identical in-frame deletion in CSF1R (CSF1RY546_K551del) in the skin lesions of both children. Recent data from murine models suggest that CSF1R-expressing yolk sac derived precursors of tissue-resident macrophages may be a cell-of-origin of the histiocytoses (Mass, et al. Nature 2017). Consistent with this hypothesis, the identical CSF1R mutation was shared across the histiocytosis lesions in both twins but was absent from blood or fingernails.We next sought to determine if similar mutations in CSF1R might exist in sporadic histiocytosis cases. We therefore sequenced 92 ECD (42%), 58 LCH (27%), 50 JXG (23%), 12 RDD (5%), and 6 histiocytic sarcoma (HS) (3%) lesions using WES, targeted DNA sequencing with a 585-gene panel, and targeted RNA-sequencing for fusions in 74 genes. This identified recurrent mutations in BRAFV600E, MAP2K1, N/KRAS, and ARAF, as well as BRAF, NTRK1, and ALK fusions as previously described in ECD and LCH (Fig. A-C). Interestingly, CSF1R mutations were also found in 8 cases, most commonly as CSF1RY546_K551del, and were predominantly in JXG (10%; n=5/50). Consistent with the recurrent nature of this mutation, expression of CSF1RY546_K551del, but not CSF1RWT, conferred robust cytokine-independent growth to cell lines normally dependent on cytokines (Ba/F3 and 32D cells; Fig. D). We also identified individuals with mutations in CSF3R, KIT, ALK, MET, JAK3, and CRAF, as well as a RET fusion. These studies additionally identified important differences in the spectrum of kinase alterations across histiocytoses subtypes. For example, the BRAFV600E mutation was the most common kinase alteration in LCH and ECD but was not identified in JXG or RDD. Furthermore, BRAF fusions were predominantly seen in LCH and JXG. Meanwhile, NTRK1 and ALK fusions were mainly identified in JXG and ECD, respectively.From a therapeutic perspective, CSF1R activating mutations sensitized cells to inhibition with the CSF1R-specific, small-molecule kinase inhibitors pexidartinib and BLZ945. In addition, in the course of this study, a patient bearing ALK-rearranged ECD required therapy, and we were able to evaluate response to the ALK inhibitor crizotinib. Crizotinib-treatment resulted in profound and sustained therapeutic improvements in this patient (Fig. E).Overall, the above data demonstrate the occurrence of activating CSF1R and other RTK alterations in patients with histiocytic neoplasms, many of which have direct therapeutic importance (such as the first demonstration of ALK inhibitor efficacy in ALK+ histiocytosis). In addition, the discovery of somatically acquired CSF1R activating mutations in identical twins with histiocytosis provides the first human evidence that tissue-resident macrophages may serve as a cell-of-origin of the histiocytoses. DisclosuresArcila:Invivoscribe, Inc.: Consultancy, Honoraria.

Resistance Mechanisms to SYK Inhibition in AML

Alterations in signaling pathways are critical to the pathogenesis of acute myeloid leukemia (AML) and are often driven by aberrant kinases. We previously identified spleen tyrosine kinase (SYK), a non-receptor, cytoplasmic tyrosine kinase, as a druggable target in AML and a critical regulator of FLT3, the most commonly mutated receptor tyrosine kinase in this disease. SYK was initially described as having an important role in B-cell development. More recent studies showed a broader relevance of SYK in hematopoietic signaling. Multiple orally bioavailable SYK inhibitors, including entospletinib (GS-9973) and TAK-659, are currently in Phase I/II clinical trials in patients with AML, with promising results. Targeted therapy, however, is typically associated with the emergence of resistance, and combination therapy is almost always needed for a durable therapeutic response. The aim of our study, therefore, was to identify resistance mechanisms to SYK inhibition in AML and to identify new synergistic drug combinations to overcome them.We conducted a genome-scale, pooled open reading frame (ORF) library screen to investigate the mechanisms of drug resistance to SYK inhibition. We screened the AML cell lines MV4;11 and MOLM-14, which are both highly sensitive to SYK inhibitors, with entospletinib after transduction with the ORF library. Expression of KRAS and HRAS ORFs, as well as other RAS signaling pathway-related proteins, emerged as conferring the strongest resistance to entospletinib in both cell lines. Upon overexpression of NRAS and KRAS in MV4;11 and MOLM-14 cells, both wildtype for RAS, we observed i) upregulation of phospho ERK 1/2, indicating activated RAS signaling, and ii) we confirmed markedly reduced sensitivity to entospletinib. We next screened 12 AML cell lines for sensitivity to entospletinib and found that the presence of activating mutations of the RAS signaling pathway was associated with innate resistance to entospletinib.To further study the mechanism of resistance to SYK inhibition, we generated entospletinib resistant cell lines by exposing MV4;11 cells to increasing concentrations of the drug for five months. In these cells, the entospletinib IC50 shifted from 500 nM to 5 μM, and these cells were also cross resistant to the SYK inhibitor PRT062607. RNA-sequencing showed increased expression of NRAS, KRAS and related pathway genes in entospletinib resistant compared to sensitive parental cells. Accordingly, gene set enrichment analysis revealed significant upregulation of RAS signaling-related gene sets. These findings were confirmed by qPCR and western blot analysis, supporting that the activation of the RAS/MAPK signaling pathway also confers acquired resistance to SYK inhibition in AML.Because RAS mutant AML cells have been reported to be sensitive to MEK inhibition, we next tested whether MEK inhibition using PD0325901 would recapture response to SYK inhibition in RAS/MAPK activated AML cells. Indeed, entospletinib was synergistic with PD0325901 in the MV4;11 entospletinib resistant cells. Moreover, entospletinib was synergistic with PD0325901 in five different AML cell lines, including AML cell lines with RAS pathway mutations. Additionally, this drug combination was synergistic when tested in a patient sample with NRAS G12D, KRAS G12D and PTPN11 G60V mutations.In conclusion, we found that RAS pathway activation results in resistance to entospletinib in AML that may be overcome with the combination of a SYK inhibitor with a MEK inhibitor. DisclosuresNo relevant conflicts of interest to declare.

Receptor Tyrosine Kinase-Targeted Cancer Therapy.

In the past two decades, several molecular targeted inhibitors have been developed and evaluated clinically to improve the survival of patients with cancer. Molecular targeted inhibitors inhibit the activities of pathogenic tyrosine kinases. Particularly, aberrant receptor tyrosine kinase (RTK) activation is a potential therapeutic target. An increased understanding of genetics, cellular biology and structural biology has led to the development of numerous important therapeutics. Pathogenic RTK mutations, deletions, translocations and amplification/over-expressions have been identified and are currently being examined for their roles in cancers. Therapies targeting RTKs are categorized as small-molecule inhibitors and monoclonal antibodies. Studies are underway to explore abnormalities in 20 types of RTK subfamilies in patients with cancer or other diseases. In this review, we describe representative RTKs important for developing cancer therapeutics and predicting or evaluated resistance mechanisms.

High-Throughput Functional Evaluation of Variants of Unknown Significance in ERBB2.

Purpose: The advent of next-generation sequencing technologies has enabled the identification of several activating mutations of Erb-B2 receptor tyrosine kinase 2 (ERBB2) among various cancers. However, the significance of infrequent mutations has not been fully investigated. Herein, we comprehensively assessed the functional significance of the ERBB2 mutations in a high-throughput manner.Experimental Design: We evaluated the transforming activities and drug sensitivities of 55 nonsynonymous ERBB2 mutations using the mixed-all-nominated-in-one (MANO) method.Results: G776V, G778_S779insG, and L841V were newly revealed to be activating mutations. Although afatinib, neratinib, and osimertinib were shown to be effective against most of the ERBB2 mutations, only osimertinib demonstrated good efficacy against L755P and L755S mutations, the most common mutations in breast cancer. In contrast, afatinib and neratinib were predicted to be more effective than other inhibitors for the A775_776insYVMA mutation, the most frequent ERBB2 mutation in lung cancer. We surveyed the prevalence of concurrent ERBB2 mutation with gene amplification and found that approximately 30% of ERBB2-amplified urothelial carcinomas simultaneously carried ERBB2 mutations, altering their sensitivity to trastuzumab, an mAb against ERBB2. Furthermore, the MANO method was applied to evaluate the functional significance of 17 compound mutations within ERBB2 reported in the COSMIC database, revealing that compound mutations involving L755S were sensitive to osimertinib but insensitive to afatinib and neratinib.Conclusions: Several ERBB2 mutations showed varying sensitivities to ERBB2-targeted inhibitors. Our comprehensive assessment of ERBB2 mutations offers a fundamental database to help customize therapy for ERBB2-driven cancers.We identified several ERBB2 mutations as activating mutations related to tumorigenesis. In addition, our comprehensive evaluation revealed that several ERBB2 mutations showed varying sensitivities to ERBB2-targeted inhibitors, and thus, the functional significance of each variant should be interpreted precisely to design the best treatment for each patient. Clin Cancer Res; 24(20); 5112-22. ©2018 AACR.

Abstract A43: Deficiency of PTPRH in pediatric solid tumors identifies a high-risk phenotype that can be overcome by combinatorial tyrosine kinase inhibition

Abstract As there is a paucity of targetable oncogenic drivers in pediatric solid tumors, we hypothesized that deficiency of select protein tyrosine phosphatases may allow proliferative signals to proceed unchecked in these cancers and that understanding these regulatory networks could inform therapy. In support of this possibility, the protein tyrosine phosphatase PTPRH has been previously reported to have a tumor-suppressive role in colon cancer and hepatocellular cancer. In the current study, we examine the role of PTPRH in pediatric solid tumors. Using multiple publicly available databases, we observed that low PTPRH expression is associated with significantly shorter survival in both neuroblastoma and osteosarcoma. Further, restoration of PTPRH expression in PTPRH-deficient neuroblastoma cells inhibits colony formation. In addition, we used a variety of biochemical approaches to identify novel PTPRH-binding partners and potential substrates. Moreover, we utilized these data to design combinatorial tyrosine kinase inhibitor therapies that target the cadre of receptor tyrosine kinases that are regulated by PTPRH. In particular, the combination of crizotinib and pazopanib inhibits proliferation of PTPRH-deficient neuroblastoma cells in vitro and reduces tumor growth in vivo. We are continuing to optimize the selection of tyrosine kinase inhibitors in order to most closely phenocopy restoration of PTPRH and maximize antitumor activity. Thus, we have shown that even for tumors that lack amplification or driver mutations of receptor tyrosine kinases, combinatorial tyrosine kinase inhibition can have substantial therapeutic activity and should be investigated further in the laboratory and the clinic. Citation Format: Kathleen A. Scorsone, Siddhartha Tyagi, Mayra C. Orellana, Sonal Gahlawat, Amritha Nair, Tingting Sun, Thomas F. Westbrook, Ronald J. Bernardi. Deficiency of PTPRH in pediatric solid tumors identifies a high-risk phenotype that can be overcome by combinatorial tyrosine kinase inhibition [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A43.

ALK-rearrangement neuroendocrine carcinoma of the lung: a comprehensive study of a rare case series and review of literature.

Driver mutations involving tyrosine kinase receptors play crucial roles in the oncogenesis of lung adenocarcinoma. However, receptor tyrosine kinase mutations are extremely rare events in primary pulmonary neuroendocrine carcinoma (NEC), which is a molecular heterogeneous entity. In this study, we examined 4 cases of NEC with anaplastic lymphoma kinase (ALK) rearrangement between 2008 and 2018 at our hospital. We comprehensively analyzed the carcinomas’ clinicopathological features, genetic alterations, and response to ALK inhibitor. One case of atypical carcinoid tumor and 1 case of large cell NEC (LCNEC) achieved response to ALK inhibitor (crizotinib) treatment. One case of combined LCNEC with adenocarcinoma harboring KLC1-ALK (K9:A20) fusion genes was confirmed by NGS of both components, while only the LCNEC component presented RB1 mutation. Notably, tumor cells of different components exhibited different ALK-positive signal patterns by fluorescence in situ hybridization, which revealed isolated 3′ signals in the adenocarcinoma component but split signals in the LCNEC. As the largest case series study, our findings suggested that preliminary screening for ALK rearrangement should also be considered in atypical carcinoid and high-grade NEC. Patients with ALK rearrangement-positive NEC would benefit from ALK inhibitor intervention.

Retrospective analysis of clinical outcome of EGFR mutated patients in a county hospital.

e21208Background: Mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase are observed in around 15% of lung adenocarcinoma in the United States. Multiple phase III trials have sho...

ERK1/2 inhibitors: New weapons to inhibit the RAS-regulated RAF-MEK1/2-ERK1/2 pathway.

The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is de-regulated in a variety of cancers due to mutations in receptor tyrosine kinases (RTKs), negative regulators of RAS (such as NF1) and core pathway components themselves (RAS, BRAF, CRAF, MEK1 or MEK2). This has driven the development of a variety of pharmaceutical agents to inhibit RAF-MEK1/2-ERK1/2 signalling in cancer and both RAF and MEK inhibitors are now approved and used in the clinic. There is now much interest in targeting at the level of ERK1/2 for a variety of reasons. First, since the pathway is linear from RAF-to-MEK-to-ERK then ERK1/2 are validated as targets per se. Second, innate resistance to RAF or MEK inhibitors involves relief of negative feedback and pathway re-activation with all signalling going through ERK1/2, validating the use of ERK inhibitors with RAF or MEK inhibitors as an up-front combination. Third, long-term acquired resistance to RAF or MEK inhibitors involves a variety of mechanisms (KRAS or BRAF amplification, MEK mutation, etc.) which re-instate ERK activity, validating the use of ERK inhibitors to forestall acquired resistance to RAF or MEK inhibitors. The first potent highly selective ERK1/2 inhibitors have now been developed and are entering clinical trials. They have one of three discrete mechanisms of action - catalytic, "dual mechanism" or covalent - which could have profound consequences for how cells respond and adapt. In this review we describe the validation of ERK1/2 as anti-cancer drug targets, consider the mechanism of action of new ERK1/2 inhibitors and how this may impact on their efficacy, anticipate factors that will determine how tumour cells respond and adapt to ERK1/2 inhibitors and consider ERK1/2 inhibitor drug combinations.

Investigating the Hetero-interactions of Receptor Tyrosine Kinases in Live Cells

Receptor tyrosine kinases (RTKs), the second largest class of membrane receptors, transduce signals across the plasma membrane via lateral association. They play an important role in development, regulating tissue growth, and controlling cell migration. Mutations in RTKs are associated with many cancers, and thus anti-RTK drugs are being actively developed and tested. While many drugs have entered the market, they largely have only been moderately successful. One possible explanation for this is a lack of biophysical understanding of how these receptors interact with each other. There is a growing body of evidence that RTKs from different subfamilies (e.g., VEGFs, Ephs, FGFRs) can form hetero-species; however, the current picture of this interactome is rather incomplete. It is possible that presently unknown interactions affect the response of a given RTK to its ligands and inhibitors. Using a quantitative FRET spectroscopy method known as Fully Quantified Spectral Imaging (FSI), we demonstrate the existence of specific interactions between members of different RTK subfamilies. Finally, we present thermodynamic models that show the relative population of heterodimers under different conditions.

Abstract A02: Succinate dehydrogenase deficiency in a PDGFRA mutated gastrointestinal stromal tumor

Abstract Most gastrointestinal stromal tumors harbor mutually exclusive gain-of-function mutations in the receptor tyrosine kinase (RTK) KIT (70-80%) or in the related receptor PDGFRA (~10%). Mutations in PDGFRA are most commonly found in the TK domain encoded by exon 18, conferring resistance to approved tyrosine kinase inhibitors (TKI). A more recently defined subset of GIST exhibit deficiencies in the succinate dehydrogenase (SDH) enzyme complex as a result of genetic or epigenetic inactivation of one of the four SDH subunit genes (SDHA-D). SDH-deficient GISTs generally lack mutations in KIT and PDGFRA and do not respond to imatinib (IM), although these GISTs may respond to TKIs targeting VEGFR, such as sunitinib and regorafenib. Whole-exome sequencing (WES) of two GISTs from a single case carrying the IM-resistant exon 18 PDGFRA mutation D842V was carried out to identify additional potential targets for therapy. Surprisingly, a somatic mutation in exon 4 of the SDHB subunit gene (c.291_292delCT, p.I97Mfs*21) was identified in both tumors. Sanger sequencing confirmed the presence of this inactivating mutation, and immunohistochemistry for the SDHB subunit demonstrated that these tumors were in fact SDH deficient. IHC for the SDHB subunit across a panel of ~100 GIST cases failed to detect SDH deficiency in other GISTs with RTK mutations. To our knowledge this is the first reported case of an SDH-deficient GIST harboring a gain-of-function mutation in the PDGFRA receptor, and this finding has implications for the molecular-based classification and treatment of GIST. Citation Format: Martin G. Belinsky, Lori Rink, Kathy K. Cai, Biao Luo, Yan Zhou, Margaret von Mehren. Succinate dehydrogenase deficiency in a PDGFRA mutated gastrointestinal stromal tumor [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A02.

Multielectrode array recordings of human iPSC-derived neurons reveal differences in network activity depending on differentiation protocol and genome modification

Event Abstract Back to Event Multielectrode array recordings of human iPSC-derived neurons reveal differences in network activity depending on differentiation protocol and genome modification Sandra K. Fischer1* and Frank Gillardon1 1 Boehringer Ingelheim (Germany), Germany With increasing prevalence of neuropsychiatric disorders, there is an ever-growing need for suitable model systems to understand the underlying pathological mechanisms. Currently most data are derived from animal models, although correlations between animal and human are often weak. However, acquisition of human neuronal material is rarely possible and afflicted with ethic concerns. Human induced pluripotent stem cell (hiPSC) derived neuronal cultures offer a possibility to overcome these limitations. Synaptic activity and neurotransmission deficits have been identified as core pathophysiologies of neuropsychiatric disorders. The multi electrode array (MEA) technology allows for repeated measurements of electrical activity of functional neuronal networks over an extended period of time. We cultivated iPSC-derived neuronal cultures on the Multichannel Systems MEA multiwell platform and assessed synaptic activity over time and in response to treatment with pharmacological agents. The hiPSC-derived neurons developed electrical activity from single spikes to bursts over time indicating maturation. Dot-seeded GlutaNeurons (iCell Neurons, Cellular Dynamics International) showed their peak activity after 2-3 weeks in culture with the highest burst activity in week 3. Ngn2-induced Neurons (iCell Neurons, Cellular Dynamics International) displayed a weaker total activity with lower spike rate, less burst activity and a maximum activity after 6 weeks in culture. Treatment with the GABA antagonist Bicuculline evoked synchronized burst firing in GlutaNeuron cultures containing approx. 20% GABAergic neurons, whereas the spontaneous activity of the purely glutamatergic culture of Induced Neurons was unchanged as expected. Loss-of-function mutations in ErbB receptor tyrosine kinases have been identified as genetic risk factors for neuropsychiatric diseases. Upon pharmacological inhibition of ErbB receptor tyrosine kinases, neuronal network activity was inhibited, consistent with our MEA recordings from primary rat cortical neuron cultures. Moreover, we generated a loss-of-function mutation in a high risk gene for schizophrenia in hiPSC from a healthy donor utilizing the CRISPR/Cas9 gene editing system. Following neuronal differentiation into the forebrain lineage electrophysiological activity was monitored on the MEA multiwell system during network formation and maturation. Preliminary data indicate altered spontaneous network activity in mutant iPSC-derived neurons compared to isogenic controls. In summary, our findings demonstrate that hiPSC-derived neurons form functional neuronal networks on MEA plates which can serve as suitable system for modeling of neuropsychiatric diseases. Acknowledgements The authors thank Dr. Sabine Lange (Cellular Dynamics International) for helpful discussions. Keywords: IPSC-derived neurons, neuropsychiatric disorder, electrical network activity, multielectrode array plate, CRISPR/Cas9 gene editing Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018. Presentation Type: Poster Presentation Topic: Stem cell-derived applications Citation: Fischer SK and Gillardon F (2019). Multielectrode array recordings of human iPSC-derived neurons reveal differences in network activity depending on differentiation protocol and genome modification. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00015 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Mrs. Sandra K Fischer, Boehringer Ingelheim (Germany), Hannover, Germany, sandra.fischer@boehringer-ingelheim.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sandra K Fischer Frank Gillardon Google Sandra K Fischer Frank Gillardon Google Scholar Sandra K Fischer Frank Gillardon PubMed Sandra K Fischer Frank Gillardon Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Abstract A159: Characterization of putative ErbB4 antagonists: targeted melanoma drug discovery

Abstract Introduction: Gain-of-function mutations in the ErbB4 receptor tyrosine kinase have been found in a significant fraction of melanoma cell lines that are dependent on ErbB4 for proliferation. However, there is a scarcity of therapeutics for treating these ErbB4-dependent tumors. Our drug discovery approach is based on the observation that the Q43L mutant of the ErbB4 agonist Neuregulin 2beta (NRG2b) functions as a partial agonist/antagonist at ErbB4. NRG2b/Q43L stimulates ErbB4 tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and competitively antagonizes agonist stimulation of ErbB4-dependent cell proliferation. ErbB4 partial agonists that function as antagonists at ErbB4 may hold promise as targeted therapeutics for ErbB4-dependent melanomas. Experimental Procedures: An automated phospho-ErbB4 sandwich ELISA and automated proliferation assays were developed and deployed to identify small-molecule compounds that are likely to function as ErbB4 partial agonists/antagonists. Hit molecules were then further evaluated for ErbB4 specificity and antiproliferative mechanism(s) of action. To examine specificity for ErbB4, we tested whether the hit molecules inhibit ErbB4-dependent and -independent proliferation of the same cell line. ErbB4 specificity was also evaluated by determining whether the hit molecules exert effects on any of the other three members of the ErbB family of receptor tyrosine kinases: EGFR, ErbB2, and ErbB3. The mechanism(s) of action for antiproliferative effects were investigated by determining whether the hit molecules inhibited known effectors that couple ErbB4 to cellular proliferation, whether they degraded ErbB4, and whether the inhibitory effects induced by the molecules were reversible. Results: High-throughput screening (HTS) strategies have been utilized to identify 20 small-molecule compounds that stimulate ErbB4 tyrosine phosphorylation, fail to stimulate ErbB4-dependent cellular proliferation, and inhibit agonist-induced ErbB4-dependent cellular proliferation. Efforts to determine whether these hits are specific for ErbB4 and by what mechanism they induce antiproliferative effects are under way and will be reported. Structures of these small-molecule ErbB4 partial agonists/antagonists may be reported, pending submission of a provisional patent application. Conclusions: Using validated HTS methodologies for identifying ErbB4 partial agonists that function as ErbB4 antagonists has led to the identification of 20 hits. Molecules that are specific for ErbB4 hold promise as targeted therapeutics for melanoma and other ErbB4-dependent tumors. Citation Format: Richard L. Cullum, Lauren M. Lucas, John T. Piazza, Jared I. Senfeld, Logan T. Neel, Ram B. Gupta, Allan E. David, David J. Riese. Characterization of putative ErbB4 antagonists: targeted melanoma drug discovery [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A159.

An Atypical Case of Guillain-Barré Syndrome Associated With Gastrointestinal Stromal Tumor

Guillain-Barré syndrome (GBS) is an acute immune-mediated polyneuropathy presenting with classic symptoms of progressive, symmetric muscle weakness and absent or depressed deep tendon reflexes. Molecular mimicry from an immune response to a proceeding infection inciting cross-reaction with peripheral nerves is the proposed etiology of GBS. The most common precipitant of GBS is Campylobacter jejuni infection. Rarely, GBS has been described to occur as a paraneoplastic syndrome with possible malignant triggers including small cell lung cancer and squamous cell carcinoma. We describe a case of GBS associated with gastrointestinal stromal tumor (GIST). To this author's knowledge, there are no other case reports in the literature of GBS associated with GIST. A 65 year-old man with a past medical history of hypertension and type II diabetes mellitus presented with 3 days of paresthesias of the fingers, toes, and mouth, slurred speech, vertigo, and mild hematemesis. His physical exam was significant for bilateral facial nerve palsy, dysarthria, and decreased reflexes in the lower extremities bilaterally. Laboratory results were significant for white blood cell count of 14 and hemoglobin (Hgb) level of 20.2. CT abdomen and pelvis with contrast showed a lobular soft tissue gastric mass 3.4 x 2.5 x 2.9 cm. CT head without contrast showed no acute intracranial process. Lumbar puncture revealed albuminocytologic dissociation. Nerve conduction study and needle electromyography showed demyelinating features. Based on these results, the patient was diagnosed with GBS, specifically the acute inflammatory demyelinating polyneuropathy variant. He was treated with 5 days of intravenous immunoglobulin (IVIG). For the gastric mass, he underwent endoscopic ultrasound-guided biopsy, and the pathology revealed GIST (Figures 1 and 2). Respiratory parameters remained stable, and he had mild improvement with IVIG. Campylobacter jejuni antibody was negative. For the elevated Hgb, the patient was negative for JAK2 V617F and CALR mutation, and erythropoietin was normal, making polycythemia vera unlikely. His Hgb trended down to within normal levels over the next month. The initial elevated Hgb could have been explained by hemoconcentration; however, there are murine models of GIST with KIT activating receptor tyrosine kinase mutations causing hematopoiesis. This case demonstrates a rare case of GBS associated with GIST. The patient is planned for open partial gastrectomy.Figure: Intramural lesion found in the lesser curvature of the stomach. It appears to originate from within the muscularis propria (Layer 4) and invades through the serosa. Fine needle aspiration initial cytology was suggestive of a spindle cell tumor. Sonographic appearance was suggestive of a GIST with concerning features (cystic components, large size, invading through serosa).Figure: EUS-FNA biopsy cytopathology images of GIST. A: Cytopathology conventional smear slide showing clusters of spindle cells typical for GIST (Papanicolaou stain, 200x); B: Cytopathology cell block preparation (H&E stain, 200x); C and D: Immunohistochemical stains performed on cell block section, positive staining of DOG-1 (C) and c-KIT (D) confirms the diagnosis of GIST (200x).

Dual Targeting of Insulin Receptor and KIT in Imatinib-Resistant Gastrointestinal Stromal Tumors.

Oncogenic KIT or PDGFRA receptor tyrosine kinase (RTK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GIST), and treatment with the KIT/PDGFRA inhibitor imatinib is the standard of care for patients with metastatic GIST. Most GISTs eventually acquire imatinib resistance due to secondary mutations in the KIT kinase domain, but it is unclear whether these genomic resistance mechanisms require other cellular adaptations to create a clinically meaningful imatinib-resistant state. Using phospho-RTK and immunoblot assays, we demonstrate activation of KIT and insulin receptor (IR) in imatinib-resistant GIST cell lines (GIST430 and GIST48) and biopsies with acquisition of KIT secondary mutations, but not in imatinib-sensitive GIST cells (GIST882 and GIST-T1). Treatment with linsitinib, a specific IR inhibitor, inhibited IR and downstream intermediates AKT, MAPK, and S6 in GIST430 and GIST48, but not in GIST882, exerting minimal effect on KIT phosphorylation in these cell lines. Additive effects showing increased apoptosis, antiproliferative effects, cell-cycle arrest, and decreased pAKT and pS6 expression, tumor growth, migration, and invasiveness were observed in imatinib-resistant GIST cells with IR activation after coordinated inhibition of IR and KIT by linsitinib (or IR shRNA) and imatinib, respectively, compared with either intervention alone. IGF2 overexpression was responsible for IR activation in imatinib-resistant GIST cells, whereas IR activation did not result from IR amplification, IR mutation, or KIT phosphorylation. Our findings suggest that combinatorial inhibition of IR and KIT warrants clinical evaluation as a novel therapeutic strategy in imatinib-resistant GISTs. Cancer Res; 77(18); 5107-17. ©2017 AACR.

Lack of microsatellite instability in gastrointestinal stromal tumors.

The microsatellite instability (MSI) phenotype may constitute an important biomarker for patient response to immunotherapy, particularly to anti-programmed death-1 inhibitors. MSI is a type of genomic instability caused by a defect in DNA mismatch repair (MMR) proteins, which is present mainly in colorectal cancer and its hereditary form, hereditary nonpolyposis colorectal cancer. Gastrointestinal stromal tumor (GIST) development is associated with activating mutations of KIT proto-oncogene receptor tyrosine kinase (KIT) or platelet-derived growth factor receptor α (PDGFRA), which are oncogenes that predict the response to imatinib mesylate. In addition to KIT/PDGFRA mutations, other molecular alterations are important in GIST development. In GISTs, the characterization of the MSI phenotype is scarce and the results are not consensual. The present study aimed to assess MSI in a series of 79 GISTs. The evaluation of MSI was performed by pentaplex polymerase chain reaction comprising five markers, followed by capillary electrophoresis. The expression of MMR proteins was evaluated by immunohistochemistry. Regarding the KIT/PDGFRA/B-Raf proto-oncogene, serine/threonine kinase molecular profile of the 79 GISTs, 83.6% of the tumors possessed KIT mutations, 10.1% had PDGFRA mutations and 6.3% were triple wild-type. The mutated-PDGFRA cases were associated with gastric location and a lower mitotic index compared with KIT-mutated and wild-types, and these patients were more likely to be alive and without cancer. MSI analysis identified 4 cases with instability in one marker, however, additional evaluation of normal tissue and immunohistochemical staining of MMR proteins confirmed their microsatellite-stable nature. The results of the present study indicated that MSI is not involved in GIST tumorigenesis and, therefore, cannot serve as a biomarker to immunotherapy response in GIST.

Succinate dehydrogenase deficiency in a PDGFRA mutated GIST

BackgroundMost gastrointestinal stromal tumors (GISTs) harbor mutually exclusive gain of function mutations in the receptor tyrosine kinase (RTK) KIT (70–80%) or in the related receptor PDGFRA (~10%). These GISTs generally respond well to therapy with the RTK inhibitor imatinib mesylate (IM), although initial response is genotype-dependent. An alternate mechanism leading to GIST oncogenesis is deficiency in the succinate dehydrogenase (SDH) enzyme complex resulting from genetic or epigenetic inactivation of one of the four SDH subunit genes (SDHA, SDHB, SDHC, SDHD, collectively referred to as SDHX). SDH loss of function is generally seen only in GIST lacking RTK mutations, and SDH-deficient GIST respond poorly to imatinib therapy.MethodsTumor and normal DNA from a GIST case carrying the IM-resistant PDGFRA D842V mutation was analyzed by whole exome sequencing (WES) to identify additional potential targets for therapy. The tumors analyzed were separate recurrences following progression on imatinib, sunitinib, and the experimental PDGFRA inhibitor crenolanib. Tumor sections from the GIST case and a panel of ~75 additional GISTs were subjected to immunohistochemistry (IHC) for the SDHB subunit.ResultsSurprisingly, a somatic, loss of function mutation in exon 4 of the SDHB subunit gene (c.291_292delCT, p.I97Mfs*21) was identified in both tumors. Sanger sequencing confirmed the presence of this inactivating mutation, and IHC for the SDHB subunit demonstrated that these tumors were SDH-deficient. IHC for the SDHB subunit across a panel of ~75 GIST cases failed to detect SDH deficiency in other GISTs with RTK mutations.ConclusionsThis is the first reported case of a PDGFRA mutant GIST exhibiting SDH-deficiency. A brief discussion of the relevant GIST literature is included.

Abstract 380: Systematic identification of novel functional tumor-specific mutations in receptor tyrosine kinases based on their pan-cancer mutational profiles in Japanese patients with cancer

Abstract Background: Recent accumulation of large amounts of cancer genome information has revealed the presence of tumor-specific mutations with unknown functions; characterization of these mutations should facilitate the identification of novel therapeutic targets. Some tumor-specific mutations in receptor tyrosine kinases (RTKs) act as oncogenic drivers in multiple cancer types. Here, we aimed to identify novel, functional, tumor-specific, single nucleotide variants (SNVs) in cancer-related RTKs based on pan-cancer mutational profiles in Japanese patients with cancer. Methods: Surgically resected tissue and corresponding peripheral blood samples of 1,685 patients enrolled in Project HOPE (High-tech Omics-based Patient Evaluation), an ongoing prospective molecular profiling study at the Shizuoka Cancer Center, collected between January 2014 and December 2015, were subjected to whole-exome sequencing with an ion torrent proton platform. Newly detected tumor-specific SNVs that could represent functional mutations between the transmembrane and kinase domains of 35 cancer-related RTKs were subjected to serum response element (SRE)-reporter assay to assess effects on extracellular signal-regulated kinase (ERK) signaling relevant to cancer cell survival. Results: We identified 201,422 nonsynonymous tumor-specific SNVs in 1,685 patients. Of these SNVs, 1,049 were observed in our focused 35 cancer-related RTKs. Approximately 45% of patients with lung squamous cell carcinoma, which was the most frequent cancer type, followed by lung adenocarcinoma (39%), harbored tumor-specific SNVs in RTKs. The relative frequencies of tumor-specific SNVs in RTKs in other tumor types were 32%, 31%, 29%, 28%, and 19% in liver cancer, colorectal cancer, gastric cancer, head and neck cancer, and breast cancer, respectively. After filtering out of SNVs registered in multiple cancer-related databases, such as COSMIC, dbSNP, and DoCM, to select putative novel tumor-specific SNVs, 22 SNVs were selected as novel tumor-specific SNVs with high potential as functional mutations through an additional selection process based on amino acid substitution patterns and multiple alignment of amino acid residues coupled with information regarding functional mutations. Among these SNVs, 13 had already been assessed using an SRE reporter assay. We identified two SNVs in NTRK1 and IGF1R that were involved in activation of the ERK pathway, indicating that these SNVs may be activating mutations. Moreover, inactivating mutations were also identified. Nine SNVs in FLT3, NTRK1, EPHA5, ERBB4, FGFR3, and KDR significantly reduced ERK activity. Conclusions: A systematic evaluation of functionally unknown tumor-specific SNVs detected in cancer genome sequencing is necessary to expand the range of molecularly targeted cancer therapeutics. Citation Format: Masakuni Serizawa, Takeshi Nagashima, Yuji Shimoda, Shumpei Ohnami, Sumiko Ohnami, Keiichi Ohshima, Tohru Mochizuki, Takashi Nakajima, Kenichi Urakami, Masatoshi Kusuhara, Ken Yamaguchi. Systematic identification of novel functional tumor-specific mutations in receptor tyrosine kinases based on their pan-cancer mutational profiles in Japanese patients with 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 380. doi:10.1158/1538-7445.AM2017-380

Abstract 4104: Intrinsic and acquired resistance to cetuximab in colorectal cancer patients

Abstract Anti-EGFR antibodies, such as cetuximab, are effective therapies for many late-stage colorectal cancer (CRC) patients; unfortunately, many tumors are initially unresponsive while others show initial efficacy but eventually develop acquired resistance. Genomic studies of patient tumors, cell lines, and xenograft models have identified putative anti-EGFR resistance markers, including mutations in KRAS, NRAS, BRAF, PIK3CA, and the EGFR extracellular domain, as well as amplifications in ERBB2 and MET. In order to further confirm and identify new resistance mechanisms to anti-EGFR treatment in CRC, we performed retrospective genomic profiling of 25 CRC patients treated at Samsung Medical Center from 2006-2015. Patients received cetuximab containing chemo regimens with varying duration of responses, including acquired resistance cases. Our analysis identifies mutations in receptor tyrosine kinases, such as EGFR, NTRK1, and PDGFRA, as well as RAS/MAPK pathway genes that affect cetuximab response. We also uncover genomic alterations in ERBB2 and c-KIT as potential novel mechanisms regulating sensitivity to anti-EGFR antibodies. Additional genomic analyses of acquired resistance tumors and in vitro studies of a patient-derived cell line provide added insights into clonal selection and signaling pathways that bypass the EGFR blockade. Overall, our study elucidates important new facets in the landscape of anti-EGFR resistance mechanisms. Note: This abstract was not presented at the meeting. Citation Format: Steven M. Bray, Jeeyun Lee, Seung Tae Kim, Philip J. Ebert, John N. Calley, Isabella H. Wulur, Thejaswini Gopalappa, Swee Seong Wong, Hui-Rong Qian, Jason C. Ting, Jiangang Liu, Melinda D. Willard, Amit Aggarwal, Ruslan D. Novosiadly, Hee-Cheol Kim, Christoph Reinhard. Intrinsic and acquired resistance to cetuximab in colorectal cancer patients [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 4104. doi:10.1158/1538-7445.AM2017-4104

Abstract 2363: Targeting Src-family kinases to combat acquired inhibitor resistance in FLT3-ITD+ AML

Abstract Activating internal tandem duplication (ITD) mutations in the FMS-like receptor tyrosine kinase 3 (Flt3) are found in approximately 30% of acute myeloid leukemia (AML) patients. Flt3-ITD expression is associated with aggressive, chemotherapy-resistant disease and decreased overall survival. Multiple selective Flt3 kinase inhibitors have been developed as targeted therapy for Flt3-ITD+ AML. However, the clinical utility of these kinase inhibitors has been limited by the rapid recurrence of drug-resistant disease that often results from Flt3 kinase domain mutations that prevent inhibitor action. Recent studies revealed that multiple non-receptor tyrosine kinases are over-expressed in AML, including the myeloid Src-family kinases Hck, Lyn and Fgr, and may cooperate with Flt3 to drive disease. These observations suggest that kinase inhibitors with specificity profiles targeting Flt3 and these AML-associated cytoplasmic kinases may be less prone to acquired resistance. In the present study, we determined the contributions of Hck and Fgr kinase activities to Flt3-ITD+ AML cell growth using a chemical genetics approach. While RNAi-mediated knockdown of each of these kinases reduces proliferation and increases apoptosis in primary AML cells, the impact of pharmacologic inhibition of their kinase activities on AML cell growth is not known. Because no selective Hck or Fgr inhibitors exist, we developed kinase domain mutants that desensitize each kinase to the broad-spectrum SRC-family kinase inhibitor, A-419259. Examination of a previous crystal structure of Hck with A-419259 bound to the active site suggested a role for the ‘gatekeeper’ residue in compound binding. Substitution of the gatekeeper threonine of Hck and Fgr with methionine conferred 3-fold resistance to A-419259 in vitro without diminishing kinase activity. When expressed in human TF-1 myeloid cells transformed with Flt3-ITD, these inhibitor-resistant Hck and Fgr mutants conferred resistance to growth inhibition by A-419259 to a similar extent, while expression of the corresponding wild-type kinases was without effect. The sensitivity of the TF-1/Flt3-ITD cell lines to A-419259 correlated with inhibition of mutant Hck and Fgr kinase activity in the cells. Our findings suggest that suppression of AML cell growth by A-419259 is due in part to inhibition of Hck and Fgr, and identify these AML-associated kinases as therapeutic targets. Ongoing studies are addressing the role of Hck and Fgr as inhibitor targets in Flt3-ITD+ AML cell lines and patient samples as well as the propensity of A-419259 and other multi-targeted inhibitors of AML-associated kinases for acquired drug resistance. Citation Format: Ravi K. Patel, Mark Weir, Sabine Hellwig, Heather Dorman, Thomas E. Smithgall. Targeting Src-family kinases to combat acquired inhibitor resistance in FLT3-ITD+ AML [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 2363. doi:10.1158/1538-7445.AM2017-2363