Role Of Receptor Tyrosine Kinases Research Articles

Platelet-derived Growth Factor Receptor-α Induces Contraction Knots and Inflammatory Pain-like Behavior in a Rat Model of Myofascial Trigger Points.

Myofascial trigger points (MTrPs) are the primary etiological characteristics of chronic myofascial pain syndrome. Receptor tyrosine kinases (RTKs) are associated with signal transduction in the central mechanisms of chronic pain, but the role of RTKs in the peripheral mechanisms of MTrPs remains unclear. The current study aimed to identify RTKs expression in MTrPs and elucidate the molecular mechanisms through which platelet-derived growth factor receptor-α (PDGFR-α) induces contraction knots and inflammatory pain-like behavior in a rat model of myofascial trigger points. MTrPs tissue samples were obtained from the trapezius muscles of patients with myofascial pain syndrome through needle biopsy, and PDGFR-α activation was analyzed by microarray, enzyme-linked immunosorbent assay, and histological staining. Sprague-Dawley rats (male and female) were used to investigate PDGFR-α signaling, assessing pain-like behaviors with Randall-Selitto and nest-building tests. Muscle fiber and sarcomere morphologies were observed using histology and electron microscopy. The PDGFR-α binding protein was identified by coimmunoprecipitation, liquid chromatograph mass spectrometer, and molecular docking. PDGFR-α-related protein or gene levels, muscle contraction, and inflammatory markers were determined by Western blot and reverse-transcription quantitative polymerase chain reaction. PDGFR-α phosphorylation levels were elevated in the MTrPs tissues of individuals with trapezius muscle pain and were positively correlated with pain intensity. In rats, PDGFR-α activation caused pain-like behaviors and muscle contraction via the Janus kinase 2/signal transducer and activator of transcription-3 (JAK2/STAT3) pathway. JAK2/STAT3 inhibitors reversed the pain-like behaviors and muscle contraction induced by PDGFR-α activation. Collagen type I α 1 (COL1A1) binds to PDGFR-α and promotes its phosphorylation, which contributed to pain-like behaviors and muscle contraction. COL1A1-induced phosphorylation of PDGFR-α and the subsequent activation of the JAK2/STAT3 pathway may induce dysfunctional muscle contraction and increased nociception at MTrPs.

Exploring Emerging Therapeutic Targets and Opportunities in Neuroendocrine Tumors: Updates on Receptor Tyrosine Kinases

Neuroendocrine tumors (NETs) represent a diverse group of neoplasms originating from neuroendocrine cells, presenting varied clinical behaviors and posing significant challenges in management. This review explores the emerging roles of receptor tyrosine kinases (RTKs) in the pathogenesis and progression of NETs, including vascular endothelial growth factor receptors (VEGFRs), insulin-like growth factor receptors (IGF-1R), RET, epidermal growth factor receptor (EGFR), and ALK. The dysregulation of RTK signaling pathways contributes to key cellular processes such as proliferation, survival, and invasion in NETs. We discuss the potential of targeting RTKs as therapeutic strategies in NETs, with a focus on recent developments in RET inhibitors and the therapeutic implications of RTK alterations.

Epidermal growth factor potentiates EGFR(Y992/1173)-mediated therapeutic response of triple negative breast cancer cells to cold atmospheric plasma-activated medium

Cold atmospheric plasma (CAP) holds promise as a cancer-specific treatment that selectively kills various types of malignant cells. We used CAP-activated media (PAM) to utilize a range of the generated short- and long-lived reactive species. Specific antibodies, small molecule inhibitors and CRISPR/Cas9 gene-editing approaches showed an essential role for receptor tyrosine kinases, especially epidermal growth factor (EGF) receptor, in mediating triple negative breast cancer (TNBC) cell responses to PAM. EGF also dramatically enhanced the sensitivity and specificity of PAM against TNBC cells. Site-specific phospho-EGFR analysis, signal transduction inhibitors and reconstitution of EGFR-depleted cells with EGFR-mutants confirmed the role of phospho-tyrosines 992/1173 and phospholipase C gamma signaling in up-regulating levels of reactive oxygen species above the apoptotic threshold. EGF-triggered EGFR activation enhanced the sensitivity and selectivity of PAM effects on TNBC cells. The proposed approach based on the synergy of CAP and EGFR-targeted therapy may provide new opportunities to improve the clinical management of TNBC.

Receptor Tyrosine Kinase: Still an Interesting Target to Inhibit the Proliferation of Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) proliferation is a critical event that contributes to the pathogenesis of vascular remodeling such as hypertension, restenosis, and pulmonary hypertension. Increasing evidences have revealed that VSMCs proliferation is associated with the activation of receptor tyrosine kinases (RTKs) by their ligands, including the insulin-like growth factor receptor (IGFR), fibroblast growth factor receptor (FGFR), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR). Moreover, some receptor tyrosinase inhibitors (TKIs) have been found and can prevent VSMCs proliferation to attenuate vascular remodeling. Therefore, this review will describe recent research progress on the role of RTKs and their inhibitors in controlling VSMCs proliferation, which helps to better understand the function of VSMCs proliferation in cardiovascular events and is beneficial for the prevention and treatment of vascular disease.

Abstract 15113: C-cbl E3 Ubiquitin Ligase Regulates Cardiomyocyte Proliferation and Cardiac Regeneration

Background: Neonatal mammalian hearts display considerable regenerative capacity following injury through cardiomyocyte proliferation. However, this regenerative capacity is lost by post-natal day 7. Previous studies have shown the role of receptor tyrosine kinases (RTKs) on cardiomyocyte proliferation and cardiac regeneration. However, the mechanisms that control RTK turnover and signaling during and beyond the regenerative window of the heart remain largely unknown. Hypothesis: This study investigates the role of the proto-oncogene Casitas b-lineage lymphoma (c-Cbl), an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets RTKs, on cardiomyocyte proliferation and cardiac regeneration following myocardial infarction (MI). Methods and Results: c-Cbl expression was downregulated during post-natal development and re-expressed in the adult heart following MI. RNA-sequencing studies reveal a distinct transcriptional profile in c-Cbl-deficient mouse hearts, of which c-Cbl deletion substantially delayed cardiomyocyte maturation and enhanced cardiomyocyte proliferation. c-Cbl deletion also extended the regenerative window for post-natal mouse hearts following MI and enhanced the ability to recover following MI with improved myocardial function, reduced scar formation, and increased cardiomyocyte cell-cycle activity. Mechanistically, c-Cbl E3 ubiquitin ligase activity was markedly enhanced in the adult than neonatal heart following MI, along with increased ubiquitination and downregulation of key RTKs involved in cardiomyocyte proliferation. Intriguingly, adenoviral expression of a c-Cbl mutant deficient in E3 ligase enhanced RTK signaling and promoted cardiomyocyte proliferation in vitro. Conclusions: These results reveal c-Cbl as a critical regulator of cardiomyocyte proliferation and a potential therapeutic target for the maintenance of cardiac regeneration and function following MI.

The two facets of receptor tyrosine kinase in cardiovascular calcification-can tyrosine kinase inhibitors benefit cardiovascular system?

Tyrosine kinase inhibitors (TKIs) are widely used in cancer treatment due to their effectiveness in cancer cell killing. However, an off-target of this agent limits its success. Cardiotoxicity-associated TKIs have been widely reported. Tyrosine kinase is involved in many regulatory processes in a cell, and it is involved in cancer formation. Recent evidence suggests the role of tyrosine kinase in cardiovascular calcification, specifically, the calcification of heart vessels and valves. Herein, we summarized the accumulating evidence of the crucial role of receptor tyrosine kinase (RTK) in cardiovascular calcification and provided the potential clinical implication of TKIs-related ectopic calcification. We found that RTKs, depending on the ligand and tissue, can induce or suppress cardiovascular calcification. Therefore, RTKs may have varying effects on ectopic calcification. Additionally, in the context of cardiovascular calcification, TKIs do not always relate to an unfavored outcome—they might offer benefits in some cases.

Receptor Tyrosine Kinases as Candidate Prognostic Biomarkers and Therapeutic Targets in Meningioma.

Meningioma (MGM) is the most common type of intracranial tumor in adults. The validation of novel prognostic biomarkers to better inform tumor stratification and clinical prognosis is urgently needed. Many molecular and cellular alterations have been described in MGM tumors over the past few years, providing a rational basis for the identification of biomarkers and therapeutic targets. The role of receptor tyrosine kinases (RTKs) as oncogenes, including those of the ErbB family of receptors, has been well established in several cancer types. Here, we review histological, molecular, and clinical evidence suggesting that RTKs, including the epidermal growth factor receptor (EGFR, ErbB1), as well as other members of the ErbB family, may be useful as biomarkers and therapeutic targets in MGM.

HER Tyrosine Kinase Family and Rhabdomyosarcoma: Role in Onset and Targeted Therapy.

Rhabdomyosarcomas (RMS) are tumors of the skeletal muscle lineage. Two main features allow for distinction between subtypes: morphology and presence/absence of a translocation between the PAX3 (or PAX7) and FOXO1 genes. The two main subtypes are fusion-positive alveolar RMS (ARMS) and fusion-negative embryonal RMS (ERMS). This review will focus on the role of receptor tyrosine kinases of the human epidermal growth factor receptor (EGFR) family that is comprised EGFR itself, HER2, HER3 and HER4 in RMS onset and the potential therapeutic targeting of receptor tyrosine kinases. EGFR is highly expressed by ERMS tumors and cell lines, in some cases contributing to tumor growth. If not mutated, HER2 is not directly involved in control of RMS cell growth but can be expressed at significant levels. A minority of ERMS carries a HER2 mutation with driving activity on tumor growth. HER3 is frequently overexpressed by RMS and can play a role in the residual myogenic differentiation ability and in resistance to signaling-directed therapy. HER family members could be exploited for therapeutic approaches in two ways: blocking the HER member (playing a driving role for tumor growth with antibodies or inhibitors) and targeting expressed HER members to vehiculate toxins or immune effectors.

Prognostic Role of Receptor Tyrosine Kinase–Like Orphan Receptors in Intestinal-Type Gastric Cancer

Background:Gastric cancer (GC) is diagnosed at advanced stages and has high mortality rates. Surgical resection and adjuvant chemotherapy are the main therapeutic approaches for GC. Despite curative resection, recurrence and metastasis contribute to a high mortality rate in patients with GC. The receptor-tyrosine-kinase-like orphan receptors 1/2 (ROR1/2) are transmembrane proteins belonging to the receptor tyrosine kinase (RTK) family. ROR1 and ROR2 are known to overexpress in the tumor tissues from several types of cancer patients. However, the role of RORs in the prognosis has not been understood. Methods:This study aimed to determine the association of mRNA expression of ROR1, ROR2, and their signaling components WNT5A, NKX2-1, and FOXF1, with the survival outcome of GC patients. We performed Kaplan-Meir survival analysis on publicly available ‘The Cancer Genome Atlas (TCGA)’ data sets using ‘Kaplan-Meir Plotter.’ Results:High mRNA expression of ROR1, ROR2, NKX2-1, and FOXF1 was significantly correlated with worse overall survival (OS) of GC patients. Interestingly ROR1 and ROR showed a prognostic role in the intestinal subtype, but not in the diffuse subtype of GC. Furthermore, ROR1 was positively correlated with regulatory T cells and M2-type macrophages and negatively correlated with Th17 and natural killer T cells in the tumor stroma of patients with GC. Conclusion:We conclude that the expression of ROR1, ROR2, and their associated genes correlate with worst prognosis of GC patients, particularly in the intestinal type.

A Computational Approach to Identify the Mutations in the Genes of the RTK Signaling Pathway and their Possible Association with Oral Squamous Cell Carcinoma

Background: We aimed to determine the role of receptor tyrosine kinases (RTK) signaling family genes in the development of oral squamous cell carcinoma (OSCC). Method: In the present in silico study, 40 whole genome sequences of patients with OSCC from the cBioPortal was analysed to identify the mutations in the genes of the RTK signaling pathway. Using the STRING v10.5, we further checked the gene with the highest frequency of mutations for its protein interactions. The obtained protein interaction network was used to identify the possible pathways related to disease phenotype. Results: Epidermal growth factor receptor (EGFR) gene showed the highest frequency of mutation (5%) among the 16 genes clustered in the RTK signaling pathway as available in the cBioportal database. Missense mutations viz., G203E, R521K were identified in the EGFR gene. The other genes which returned positive results during analysis were ERBB4 (D245N, L993S), PDGFB (R100H), and PDGFRB (L667M). Conclusion: The in silico method of analysis can be a contemporary approach for identifying possible mutations or pathways associated with the development of OSCC. Further high throughput strategies should be applied to substantiate the role of the genes identified in the present study and draw conclusive evidence as to their association with the disease phenotype.

The Role of Receptor Tyrosine Kinases in Lassa Virus Cell Entry

The zoonotic Old World mammarenavirus Lassa (LASV) causes severe hemorrhagic fever with high mortality and morbidity in humans in endemic regions. The development of effective strategies to combat LASV infections is of high priority, given the lack of a licensed vaccine and restriction on available treatment to off-label use of ribavirin. A better understanding of the fundamental aspects of the virus’s life cycle would help to improve the development of novel therapeutic approaches. Host cell entry and restriction factors represent major barriers for emerging viruses and are promising targets for therapeutic intervention. In addition to the LASV main receptor, the extracellular matrix molecule dystroglycan (DG), the phosphatidylserine-binding receptors of the Tyro3/Axl/Mer (TAM), and T cell immunoglobulin and mucin receptor (TIM) families are potential alternative receptors of LASV infection. Therefore, the relative contributions of candidate receptors to LASV entry into a particular human cell type are a complex function of receptor expression and functional DG availability. Here, we describe the role of two receptor tyrosine kinases (RTKs), Axl and hepatocyte growth factor receptor (HGFR), in the presence and absence of glycosylated DG for LASV entry. We found that both RTKs participated in the macropinocytosis-related LASV entry and, regardless of the presence or absence of functional DG, their inhibition resulted in a significant antiviral effect.

Roles for receptor tyrosine kinases in tumor progression and implications for cancer treatment.

Growth factors and their receptor tyrosine kinases (RTKs), a group of transmembrane molecules harboring cytoplasm-facing tyrosine-specific kinase functions, play essential roles in migration of multipotent cell populations and rapid proliferation of stem cells' descendants, transit amplifying cells, during embryogenesis and tissue repair. These intrinsic functions are aberrantly harnessed when cancer cells undergo intertwined phases of cell migration and proliferation during cancer progression. For example, by means of clonal expansion growth factors fixate the rarely occurring driver mutations, which initiate tumors. Likewise, autocrine and stromal growth factors propel angiogenesis and penetration into the newly sprouted vessels, which enable seeding micro-metastases at distant organs. We review genetic and other mechanisms that preempt ligand-mediated activation of RTKs, thereby supporting sustained cancer progression. The widespread occurrence of aberrant RTKs and downstream signaling pathways in cancer, identifies molecular targets suitable for pharmacological intervention. We list all clinically approved cancer drugs that specifically intercept oncogenic RTKs. These are mainly tyrosine kinase inhibitors and monoclonal antibodies, which can inhibit cancer but inevitably become progressively less effective due to adaptive rewiring processes or emergence of new mutations, processes we overview. Similarly important are patient treatments making use of radiation, chemotherapeutic agents and immune checkpoint inhibitors. The many interfaces linking RTK-targeted therapies and these systemic or local regimens are described in details because of the great promise offered by combining pharmacological modalities.

STEM-06. TISSUE FACTOR SIGNALING IN GLIOBLASTOMA TUMOR-INITIATING CELLS

Abstract Glioblastoma (GBM) is an aggressive form of brain cancer with few treatment options, characterized by over-activation of receptor tyrosine kinases (RTKs). Brain tumor-initiating cells (BTICs) is a subpopulation of cells in GBM which have the unique capacity to self-renew, facilitating recurrence. Tissue Factor (TF), normally involved in blood coagulation, is elevated in GBM and strongly correlates with more aggressive behavior. We hypothesize that TF promotes malignant behavior in glioblastoma by activating protease-activated receptor 2 (PAR2), leading to RTK stimulation and the maintenance of brain tumor-initiating cells. Therefore, our approaches are to determine: the mechanisms by which TF-PAR2 stimulates RTK signaling in GBM; the role of RTKs in mediating TF-induced BTIC maintenance in GBM, and the effect of blocking TF signaling on RTK-based therapies against GBM. Our data indicate that TF can activate multiple classes of tumor-promoting RTKs, and sustains cell proliferation through its main receptor, PAR2, even in the presence of an RTK inhibitor. We also found that TF suppression impairs the BTIC phenotype in high TF-expressing, RTK-driven GBM. This project will advance our understanding of how TF promotes malignant behavior, identify a powerful means by which cancers can resist RTK inhibitors currently in clinical use, and demonstrate the therapeutic potential of blocking TF-PAR2 signaling in cancer.

Influenza virus entry via the GM3 ganglioside-mediated platelet-derived growth factor receptor β signalling pathway.

The possible resistance of influenza virus against existing antiviral drugs calls for new therapeutic concepts. One appealing strategy is to inhibit virus entry, in particular at the stage of internalization. This requires a better understanding of virus-host interactions during the entry process, including the role of receptor tyrosine kinases (RTKs). To search for cellular targets, we evaluated a panel of 276 protein kinase inhibitors in a multicycle antiviral assay in Madin-Darby canine kidney cells. The RTK inhibitor Ki8751 displayed robust anti-influenza A and B virus activity and was selected for mechanistic investigations. Ki8751 efficiently disrupted the endocytic process of influenza virus in different cell lines carrying platelet-derived growth factor receptor β (PDGFRβ), an RTK that is known to act at GM3 ganglioside-positive lipid rafts. The more efficient virus entry in CHO-K1 cells compared to the wild-type ancestor (CHO-wt) cells indicated a positive effect of GM3, which is abundant in CHO-K1 but not in CHO-wt cells. Entering virus localized to GM3-positive lipid rafts and the PDGFRβ-containing endosomal compartment. PDGFRβ/GM3-dependent virus internalization involved PDGFRβ phosphorylation, which was potently inhibited by Ki8751, and desialylation of activated PDGFRβ by the viral neuraminidase. Virus uptake coincided with strong activation of the Raf/MEK/Erk cascade, but not of PI3K/Akt or phospholipase C-γ. We conclude that influenza virus efficiently hijacks the GM3-enhanced PDGFRβ signalling pathway for cell penetration, providing an opportunity for host cell-targeting antiviral intervention.

Clinical Evidence on the Magnitude of Change in Growth Pathway Activity in Relation to Tamoxifen Resistance is Required.

Despite prolonged disease-free survival and overall survival rates in Estrogen Receptor (ER)-positive patients undergoing adjuvant treatment, Tamoxifen therapy tends to fail due to eventual acquisition of resistance. Although numerous studies have emphasized the Role of Receptor Tyrosine Kinases (RTKs) in the development of Tamoxifen resistance, inadequate clinical evidence is available regarding the alteration of biomarker expression during acquired resistance, thus undermining the validity of the findings. Results of two meta-analyses investigating the effect of HER2 status on the prognosis of Tamoxifen-receiving patients have demonstrated that despite HER2-negative patients having longer disease-free survival; there is no difference in overhaul survival between the two groups. Furthermore, due to the intricate molecular interactions among estrogen receptors including ERα36, ERα66, and also RTKs, it is not surprising that RTK suppression does not restore Tamoxifen sensitivity. In considering such a complex network, we speculate that by the time HER2/EGFR is suppressed via targeted therapies, activation of ERα66 and ERα36 initiate molecular signaling pathways downstream of RTKs, thereby enhancing cell proliferation even in the presence of both Tamoxifen and RTK inhibitors. Although clinical findings regarding the molecular pathways downstream of RTKs have been thoroughly discussed in this review, further clinical studies are required in determining a consistency between preclinical and clinical findings. Discovering the best targets in preventing tumor progression requires thorough comprehension of estrogen-dependent and estrogen-independent pathways during Tamoxifen resistance development. Indeed, exploring additional clinically-proven targets would allow for better characterized treatments being available for breast cancer patients.

Abstract 3205: Histone tyrosine phosphorylation determines glioblastoma cell survival

Abstract Histone modification is crucial for cell signaling pathways and regulates predominantly chromatin functions related with DNA damage response (DDR) that lead cancer cells to survive upon different therapies. Receptor tyrosine kinases (RTKs) have been extensively studied in cancer progression and DDR and, recently, several reports described the role of RTKs in histone tyrosine modification. We have been focusing on the role of the receptor tyrosine kinase, TIE2, in gliomas. Our study identified the expression of TIE2 in human surgical glioma specimens in relation to malignancy, specifically in brain tumor stem cells. We have revealed that the unexpected membrane-to-nuclear trafficking of TIE2 is related with radioresistance of brain tumor stem cells, functioning as a genotoxic stress sensor to activate DNA repair machinery. Interestingly, TIE2 binds, upon IR stress, to DNA/protein complexes and phosphorylates several core histones. The tandem mass spectrum of H4 and H2B demonstrates that TIE2 directly phosphorylates not previously described H4pY51 and H2BY40 respectively, which reinvigorates TIE2's role in histone code modification. We screened for pY-readers using a SH2 domain array and found that H4pY51 and H2pY40 modifications are read by the proto-oncogenes ABL1 and CRK, respectively. Our data also showed that TIE2/H4pY54/ABL1 complex binds to DNA repair proteins, activating an NHEJ DNA repair mechanism. We summarize that upon IR stress TIE2 localizes to the nucleus where it is involved in key cellular functions by directly phosphorylating core histones, and recruiting SH2 domain proteins to the DNA damage sites, complexing to the DNA repair machinery, which in turns results in radioresistance. Our data highlight the role of epigenetics in modulating the DNA damage response of glioma radiation therapy and open new avenues to find novel targetable pathways for treatment of patients with glioblastomas. Citation Format: Mohammad Belayat Hossain, Rehnuma Shifat, Debora K. Kim, Yiesel Rivera-Molina, Francisco Puerta Martinez, Hong Jiang, Teresa Nguyen, David G. Johnson, Mark T. Bedford, Mien-Chie Hung, Erik P. Sulman, Frederick Lang, Raymond Sawaya, Juan Fueyo, Candelaria Gomez-Manzano. Histone tyrosine phosphorylation determines glioblastoma cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3205.

Abstract 4291: Oncogenic ALK regulates cell cycle progression via CDK1: Implications for therapy

Abstract Pharmacologic inhibition of oncogenic ALK can yield clinical responses in a variety of cancer types, including non-small cell lung cancer, anaplastic large-cell lymphoma, and neuroblastoma; however, de novo and acquired resistance remain major problems. As is seen with inhibition of other oncogenic receptor tyrosine kinases, ALK inhibition primarily induces G1 cell cycle arrest. However, we and others have also observed a role for receptor tyrosine kinases in regulating G2/M progression. For instance, oncogenic HER2 has been shown to directly phosphorylate tyrosine 15 of CDK1, the mitosis promoting factor. This tyrosine is also the well-known substrate of WEE1 and serves to inactivate CDK1 kinase activity as a critical component of the G2-M checkpoint. In the current study, we aimed to determine if oncogenic ALK functions in a similar capacity. We used recombinant proteins to demonstrate that ALK is also capable of directly phosphorylating tyrosine 15 of CDK1. Further, combined inhibition of ALK and WEE1 with crizotinib and AZD-1775 leads to greater reduction in pY15-CDK1 than either agent alone in ALK-amplified neuroblastoma cells. These findings suggest a model where oncogenic ALK and WEE1 provide redundant regulation of the G2-M checkpoint, which may be critical in cancer cells that have lost some of the usual complement of checkpoint controls, such as p53. Consequently, either ALK or WEE1 activity would be sufficient to maintain appropriate cell cycle regulation, while dual blockade would allow mitosis to proceed unchecked. In agreement with these predictions, we performed live cell imaging and observed that combined inhibition of ALK and WEE1 leads to aberrant mitotic progression and mitotic catastrophe. Further, this combination yields greater anti-proliferative activity than either agent alone in ALK-driven models. Thus, this therapeutic strategy holds great promise for ALK-driven cancers. Citation Format: Kathleen A. Scorsone, Sonal Gahlawat, Siddhartha Tyagi, Mayra C. Orellana, Thomas F. Westbrook, Ronald J. Bernardi. Oncogenic ALK regulates cell cycle progression via CDK1: Implications for therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4291.

Next-generation sequencing analysis of receptor-type tyrosine kinase genes in surgically resected colon cancer: identification of gain-of-function mutations in the RET proto-oncogene

BackgroundImprovement in genetic characterization of Colon Cancer (CC) patients is required to propose new potential targets, since surgical resection coupled to chemotherapy, presents several limits such as cancer recurrence and drug resistance. Targeted therapies have more efficacy and less toxicity than standard treatments. One of the most relevant cancer-specific actionable targets are receptor tyrosine kinases (RTKs) whose role in CC need to be better investigated.MethodsWe have analysed 37 CC patients using the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP). We have confirmed the somatic nature of RET variants through Sanger sequencing and assessed RET activation status and protein expression by immunofluorescence and western-blot analyses. We have used RET mutant expression vectors to evaluate the effect of selected mutations in HEK293 cells by performing proliferation, migration and clonogenic assays.ResultsAmong the 409 cancer-related genes included in the CCP we have focused on the RTKs. Overall, we have observed 101 different potentially damaging variants distributed across 31 RTK genes in 28 patients. The most frequently mutated RTKs were FLT4, ROS1, EPH7, ERBB2, EGFR, RET, FGFR3 and FGFR4. In particular, we have identified 4 different somatic variants in 10% of CC patients in RET proto-oncogene. Among them, we have demonstrated that the G533C variant was able to activate RET by promoting dimer formation and enhancing Y1062 phosphorylation. Moreover, we have demonstrated that RET G533C variant was able to stimulate anchorage-dependent proliferation, migration and clonogenic cell survival. Notably, the effects induced by the RET G533C variant were abolished by vandetanib.ConclusionsThe discovery of pathogenic variants across RTK genes in 75% of the CC patients under analysis, suggests a previously underestimated role for RTKs in CC development. The identification of a gain-of-function RET mutation in CC highlights the potential use of RET in targeted therapy.

The Origins and Vulnerabilities of Two Transmissible Cancers in Tasmanian Devils

SummaryTransmissible cancers are clonal lineages that spread through populations via contagious cancer cells. Although rare in nature, two facial tumor clones affect Tasmanian devils. Here we perform comparative genetic and functional characterization of these lineages. The two cancers have similar patterns of mutation and show no evidence of exposure to exogenous mutagens or viruses. Genes encoding PDGF receptors have copy number gains and are present on extrachromosomal double minutes. Drug screening indicates causative roles for receptor tyrosine kinases and sensitivity to inhibitors of DNA repair. Y chromosome loss from a male clone infecting a female host suggests immunoediting. These results imply that Tasmanian devils may have inherent susceptibility to transmissible cancers and present a suite of therapeutic compounds for use in conservation.

Abstract 2086: Dominant role of receptor tyrosine kinase in the activation of MAPK signaling in BRAF non-V600 mutant cancers

Abstract BRAF mutations are found in ~8% of all cancers. In addition to the commonest mutation in kinase domain at the V600, a wide range of other missense mutations (non-V600) have been reported. Non-V600 mutant BRAF protein was also shown to enhance MAPK signaling, suggesting the effectiveness of MEK inhibitors. However, the existence of feedback mechanism in MAPK singling and the roles of receptor tyrosine kinases (RTKs) in these cells are not fully understood. To interrogate this, BRAF non-V600 cell lines with intermediate/impaired kinase activity were treated with MEK inhibitor. RTKs involved in the upregulation of P-MEK following MEK inhibition in BRAF non-V600E mutant cell lines were determined by phosphor-RTK arrays. The effect of combinatorial inhibition of RTKs and MEK in BRAF non-V600 cancer was assessed in vitro and in vivo. By analyzing drug screen data, we first found cells with BRAF non-V600E mutation were less sensitive to MEK inhibition compared with BRAF V600E mutant cell lines and almost comparable to RAS/RAF wild-type cell lines. MEK inhibition did not induce P-MEK suppression and it resulted in significant upregulation of p-MEK compared to BRAF V600E mutant cell lines. Phospho-RTK arrays in the presence or absence of trametinib displayed basal phosphorylation of RTKs including EGFR, MET, and IGF1R, although the phosphorylation level was not consistently changed following MEK inhibition. Among these RTKs, treatment with the EGFR inhibitor erlotinib led to more complete suppression of P-ERK upon trametinib treatment. Furthermore, we found that erlotinib monotherapy downregulated P-ERK in BRAF non-V600 mutant cell lines in contrast that inhibition of RTKs had no effect on MAPK signaling in BRAF V600 mutant cells. While erlotinib treatment led to MAPK inhibition both in intermediate active and impaired kinase active BRAF mutant cells, p-MEK and p-ERK were more strongly suppressed in impaired type BRAF non-V600 mutant cells. The combination of EGFR inhibitor and MEK inhibitor suppressed cell growth and achieved tumor shrinkage in mice xenograft. These results suggest that RTKs, especially EGFR, are dominantly involved in the regulation of MAPK signaling in BRAF non-V600 cancer cells. Note: This abstract was not presented at the meeting. Citation Format: Hiromichi Ebi, Hiroshi Kotani, Hidenori Kitai, Yuta Adachi, Seiji Yano. Dominant role of receptor tyrosine kinase in the activation of MAPK signaling in BRAF non-V600 mutant cancers [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 2086. doi:10.1158/1538-7445.AM2017-2086