EGFR blockade leads to singular oncogene-addiction in ETV6-NTRK3 transformed human epithelial cells and hypersensitization to entrectinib.

e18055 Background: Entrectinib, a TRK kinase inhibitor, has been approved for the treatment of tissue agnostic rare tumors positive for TRK fusions. A very low frequency molecular subset of TRK fusion tumors dubbed as secretory carcinoma (SC) are characterized by organ-agnostic epithelial origin, ETV6-NTRK3 (EN) fusion and distinguishable secretory-type tumor cells. These tumors have been frequently miscategorized as acinic cell carcinoma or adenocarcinoma. A previous mouse study identified EGF dependent epithelial progenitors as putative cell-of-origin for SC. Methods: To test the role of EGFR signaling in EN mediated transformation and therapy resistance, we expressed EN, kinase-dead EN-K380M, and drug-resistant EN-G623R in human epithelial MCF10A cells with EGF-dependent primitive function and investigated their ability to grow in the presence and absence of EGF and/or entrectinib. To understand the significance of findings based on our model, we analyzed a total of 22 ‘rare’ patients from Mayo Clinic Tissue Registry and analyzed TCGA PanCan datasets. Results: We report herein that EGF signaling is essential for normal growth but dispensable during EN driven transformation. Our findings suggest that levels equivalent to circulating EGF (0.5-1ng/ml) is sufficient to drive 100% resistance to entrectinib in vitro. Three different strategies to blockade EGF/EGFR axis including depletion of EGF in culture system, genetic depletion of EGFR using shRNA as well as cetuximab antibody-based EGFR neutralization potentiated oncogene-addiction and hypersensitivity to entrectinib in our models. As predicted, models with G623R mutation in EN was refractory to entrectinib under all experimental conditions. Further omics analysis of TCGA PanCan suggested that EN and EGFR mutations are mutually exclusive and entrectinib-resistant G623R mutation were uncommon. Interestingly, nearly all EN tumors from Mayo Clinic Tissue Registry immunostained weakly or strongly for EGFR and showed perfect concordance with pEGFR suggesting pathway activation. Conclusions: Together, these findings raise an important question whether blockade of ‘wildtype’ EGFR signaling could improve medical intervention in SC patients presenting with wildtype EGFR and no drug-resistant mutation in entrectinib, by improving oncogene-addiction and attendant hypersensitization of transformed cells to entrectinib.

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Transfer of Extracellular Vesicle-Associated-RNAs Induces Drug Resistance in ALK-Translocated Lung Adenocarcinoma

Cancers ◽

10.3390/cancers11010104 ◽

2019 ◽

Vol 11 (1) ◽

pp. 104 ◽

Cited By ~ 6

Author(s):

Hoi-Hin Kwok ◽

Ziyu Ning ◽

Peony Chong ◽

Thomas Wan ◽

Margaret Ng ◽

...

Keyword(s):

Drug Resistance ◽

Lung Adenocarcinoma ◽

Kinase Inhibitor ◽

Quantitative Polymerase Chain Reaction ◽

Tumour Microenvironment ◽

Extracellular Vesicle ◽

Drug Resistant ◽

Anaplastic Lymphoma ◽

Tki Treatment

Anaplastic lymphoma kinase (ALK) translocation is an actionable mutation in lung adenocarcinoma. Nonetheless tumour consists of heterogeneous cell subpopulations with diverse phenotypes and genotypes, and cancer cells can actively release extracellular vesicles (EVs) to modulate the phenotype of other cells in the tumour microenvironment. We hypothesized that EVs derived from a drug-resistant subpopulation of cells could induce drug resistance in recipient cells. We have established ALK-translocated lung adenocarcinoma cell lines and subclones. The subclones have been characterized and the expression of EV-RNAs determined by quantitative polymerase chain reaction. The effects of EV transfer on drug resistance were examined in vitro. Serum EV-RNA was assayed serially in two patients prescribed ALK-tyrosine kinase inhibitor (ALK-TKI) treatment. We demonstrated that the EVs from an ALK-TKI-resistant subclone could induce drug resistance in the originally sensitive subclone. EV-RNA profiling revealed that miRNAs miR-21-5p and miR-486-3p, and lncRNAs MEG3 and XIST were differentially expressed in the EVs secreted by the resistant subclones. These circulating EV-RNA levels have been found to correlate with disease progression of EML4-ALK-translocated lung adenocarcinoma in patients prescribed ALK-TKI treatment. The results from this study suggest that EVs released by a drug-resistant subpopulation can induce drug resistance in other subpopulations and may sustain intratumoural heterogeneity.

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Hyaluronic acid-functionalized gelatin hydrogels reveal extracellular matrix signals temper the efficacy of erlotinib against patient-derived glioblastoma specimens

10.1101/556324 ◽

2019 ◽

Author(s):

Sara Pedron ◽

Gabrielle L. Wolter ◽

Jee-Wei E. Chen ◽

Sarah E. Laken ◽

Jann N. Sarkaria ◽

...

Keyword(s):

Extracellular Matrix ◽

Hyaluronic Acid ◽

Tumor Microenvironment ◽

Kinase Inhibitor ◽

Egfr Mutations ◽

Primary Glioblastoma ◽

Stat3 Phosphorylation

AbstractTherapeutic options to treat primary glioblastoma (GBM) tumors are scarce. GBM tumors with epidermal growth factor receptor (EGFR) mutations, in particular a constitutively active EGFRvIII mutant, have extremely poor clinical outcomes. GBM tumors with concurrent EGFR amplification and active phosphatase and tensin homolog (PTEN) are sensitive to the tyrosine kinase inhibitor erlotinib, but the effect is not durable. A persistent challenge to improved treatment is the poorly understood role of cellular, metabolic, and biophysical signals from the GBM tumor microenvironment on therapeutic efficacy and acquired resistance. The intractable nature of studying GBM cell in vivo motivates tissue engineering approaches to replicate aspects of the complex GBM tumor microenvironment. Here, we profile the effect of erlotinib on two patient-derived GBM specimens: EGFR+ GBM12 and EGFRvIII GBM6. We use a three-dimensional gelatin hydrogel to present brain-mimetic hyaluronic acid (HA) and evaluate the coordinated influence of extracellular matrix signals and EGFR mutation status on GBM cell migration, survival and proliferation, as well as signaling pathway activation in response to cyclic erlotinib exposure. Comparable to results observed in vivo for xenograft tumors, erlotinib exposure is not cytotoxic for GBM6 EGFRvIII specimens. We also identify a role of extracellular HA (via CD44) in altering the effect of erlotinib in GBM EGFR+ cells by modifying STAT3 phosphorylation status. Taken together, we report an in vitro tissue engineered platform to monitor signaling associated with poor response to targeted inhibitors in GBM.

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Targeting Drug-Resistant CML and Ph+-ALL with the Spectrum Selective Protein Kinase Inhibitor XL228.

Blood ◽

10.1182/blood.v110.11.474.474 ◽

2007 ◽

Vol 110 (11) ◽

pp. 474-474 ◽

Cited By ~ 7

Author(s):

Neil P. Shah ◽

Corynn Kasap ◽

Ronald Paquette ◽

Jorge Cortes ◽

Javier Pinilla ◽

...

Keyword(s):

Protein Kinase ◽

Kinase Inhibitor ◽

Plasma Concentrations ◽

Protein Kinase Inhibitor ◽

Myelogenous Leukemia ◽

Drug Resistant ◽

Wild Type ◽

Inhibition Of Proliferation ◽

Abl Kinase

Abstract The fusion protein BCR-ABL is a hallmark of chronic myelogenous leukemia (CML) and Philadelphia-positive acute lymphocytic leukemia (Ph+-ALL), and has been demonstrated as the primary driver of these diseases. Control of CML for considerable periods of time has been achieved through use of selective ABL kinase inhibitors, particularly imatinib. Once patients fail imatinib therapy, they are commonly found to harbor a mutated and activated form of BCR-ABL which is unable to bind the inhibitor. A more recent ABL inhibitor, dasatinib, can block growth of cells harboring most of the imatinib-resistant mutations, but T315I and F317L mutations are often seen in patients relapsing after dasatinib therapy. Thus once a patient develops CML harboring these mutations, there are few therapeutic options available. XL228 is a potent multi-targeted protein kinase inhibitor with activity against IGF1R, src, and Abl. It displays low nanomolar biochemical activity against wild type Abl kinase (Ki = 5 nM), as well as the T315I form of Abl resistant to imatinib and dasatinib (Ki = 1.4 nM). XL228 also inhibits Aurora A with an IC50 of approximately 3 nM, demonstrating a more balanced inhibition profile compared to other dual Abl /Aurora inhibitors. CML and ALL cell lines were evaluated for sensitivity to XL228, and in each case the IC50 for inhibition of proliferation was less than 100 nM. XL228 inhibits phosphorylation of BCR-ABL and its substrate STAT5 in K562 cells in vitro with IC50s of 33 and 43 nM, respectively. Single-dose pharmacodynamics studies demonstrate a potent effect of XL228 on BCR-ABL signaling in K562 xenograft tumors. Phosphorylation of BCR-ABL was decreased by 50% at XL228 plasma concentrations of 3.5 μM; a similar decrease in phospho-STAT5 occurred at 0.8 μM plasma concentration. XL228 showed clear superiority to MK-0457, imatinib, and dasatinib in downregulating BCR-ABL phosphorylation in BaF3 cells expressing the T315I form of BCR-ABL in vitro (406 nM, 6912 nM, >10,000 nM, >10,000 nM, respectively), and in xenograft experiments in vivo. These results indicate that XL228 potently inhibits wild type and T315I forms of BCR-ABL, and provide a rationale for the clinical development of this agent for the treatment of patients with drug-resistant disease. A phase I dose escalation study of XL228 in subjects with CML or Ph+-ALL who have failed prior imatinib and dasatinib therapy has been initiated, focusing on safety/tolerability, pharmacodynamics, and pharmacokinetics. Pharmacodynamic assessments include a flow cytometry-based phospho-CrkL assay, quantitative PCR for BCR-ABL, and plasma markers of XL228 activity. An update on our clinical experience with XL228 in subjects resistant or intolerant to imatinib and dasatinib will be presented.

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Efficacy of gefitinib and radiotherapy combination in Indonesian patients with lung adenocarcinoma

Romanian Journal of Internal Medicine ◽

10.2478/rjim-2018-0011 ◽

2018 ◽

Vol 56 (3) ◽

pp. 173-181

Author(s):

Elisna Syahruddin ◽

Aida Lufti Huswatun ◽

Ari Prabowo ◽

Jamal Zaini ◽

Fariz Nurwidya ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Point Mutation ◽

Egfr Mutation ◽

Kinase Inhibitor ◽

Superior Vena ◽

Vena Cava ◽

Egfr Mutations ◽

Radio Therapy ◽

Egfr Mutant

Abstract Introduction. Combinations of gefitinib and radiotherapy have been observed to have synergistic and anti-proliferative effects on lung cancer in vitro. In the clinical setting, patients who presented with respiratory difficulties such as superior vena cava syndrome (SVCS), radiotherapy should be given immediately to address the emergency while waiting for the results of epidermal growth factor receptor (EGFR) mutation test. However, there has been no study that described the role of radio-therapy in Indonesian patients with EGFR-mutant lung adenocarcinoma. Methods. This preliminary study aimed to evaluate the efficacy and toxicities of gefitinib and radiotherapy combination in lung adenocarcinoma patients in Persahabatan National Respiratory Referral Hospital, Jakarta, Indonesia. Subjects were consecutively recruited between January 2013 and December 2016. Results. Thirty-one lung adenocarcinoma with EGFR mutations were enrolled. Most of them were male (51.61%) with a median age of 54.5 years old (range 38-70 years old). EGFR mutation characteristics were on exon 21 L858R point mutation (61.30%), exon 21 L861Q point mutation (16.12%) and exon 19 deletion (22.58%). Radiotherapy was given at doses between 30-60 Gy. Among these subjects, median progression-free survival (PFS) was 185 days (95%CI; 123.69 – 246.30), 1-year survival rate (1-yr) was 45.2%, and median overall survival (OS) was 300 days (95%CI; 130.94 – 469.06). There were no grade 3/4 hematological and nonhematological toxicities recorded. The most frequent grade 1 and 2 non-hematological toxicities were skin rash, diarrhea, and paronychia that might be related to tyrosine kinase inhibitor (TKI). Conclusion. The combination of TKI with radiation may be considered in EGFR-mutant lung adenocarcinoma subjects.

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Survival, Virulence Gene Expression and Difference in the Drug Susceptibility of Non-typhoidal Salmonella in Gut Physiological Conditions

10.21203/rs.3.rs-479102/v1 ◽

2021 ◽

Author(s):

Akshatha Kotian ◽

Vankadari Aditya ◽

Jassiya Sheikh ◽

Sreya Saikrishnan ◽

Praveen Rai ◽

...

Keyword(s):

Gene Expression ◽

Virulence Gene ◽

Survival Strategies ◽

Resistance Pattern ◽

Drug Resistant ◽

Experimental Conditions ◽

Virulence Gene Expression ◽

Physiological Conditions ◽

Significant Difference

Abstract Salmonella is one among the most versatile and resilient enteric pathogens that known to have developed various survival strategies within the host system. The ability of the bacteria to circumvent the physiological parameters as well as dodge the antimicrobial stress environment within the host is one of the most crucial steps in establishing an infection. With an alarming rise in multi-drug resistant serovars of non-typhoidal Salmonella and lack of vaccine for combatting the infections, behaviour of the bacteria in the presence of host physiological gut conditions (NaCl, high and low iron) and antibiotics will help in understanding the survival strategies as well as mechanisms of resistance. 59 non-typhoidal Salmonella serovars isolated from poultry and seafood were used in the study. The isolates were screened for their antibiotic susceptibility patterns. Two multi-drug resistant and two sensitive serovars were used for growth kinetics and virulence gene expression study. The results obtained revealed that despite similar resistance pattern, the effect of individual class of antibiotics on the growth of serovars varied. On contrary, no significant difference was observed in growth pattern on exposure to in vitro gut like experimental conditions. Nevertheless, the in-vitro gut conditions and exposure to antibiotics have drastically reduced the minimum inhibitory concentration of antibiotics in resistant strains. A first of its kind study that draws attention on the significant effect of antibiotics and gut physiological conditions on MIC and expression of virulence genes from Salmonella pathogenicity island (SPI) 1 and 2 between resistant and sensitive non typhoidal Salmonella serovars.

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Rho-associated Protein Kinase 2 Confers Epileptogenesis through the Activation of Astroglial Stat3 Pathway

10.21203/rs.3.rs-115889/v1 ◽

2020 ◽

Author(s):

Li-jia Song ◽

Hua Zhang ◽

Jun-gong Jin ◽

Chao Wang ◽

Xiao-Peng Qu ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Kinase Inhibitor ◽

Expression Profiles ◽

Control Group ◽

Drug Resistant ◽

Cycle Progression ◽

Stat3 Pathway

Abstract Patients with temporal lobe epilepsy (TLE) are prone to tolerance to antiepileptic drugs. Based on the perspective of molecular targets for drug resistance, it is necessary to explore effective drug resistant genes and signaling pathways for the treatment of TLE. We performed gene expression profiles in hippocampus of patients with drug-resistant TLE and identified ROCK2 as one of the 20 most significantly increased genes in hippocampus. In vitro and in vivo experiments were performed to identify the potential role of ROCK2 in epileptogenesis. In addition, the activity of Stat3 pathway was tested in hippocampal tissues and primary cultured astrocytes. The expression levels of ROCK2 in the hippocampus of TLE patients were significantly increased compared with the control group, which was due to the hypomethylation of ROCK2 promoter. Fasudil, a specific Rho-kinase inhibitor, alleviated epileptic seizures in the pilocarpine rat model of TLE. Furthermore, ROCK2 activated the Stat3 pathway in pilocarpine-treated epilepsy rats, and the spearman correlation method confirmed that ROCK2 is associated with Stat3 activation in TLE patients. In addition, ROCK2 was predominantly expressed in astrocytes during epileptogenesis, and induced epileptogenesis by activating astrocyte cell cycle progression via Stat3 pathway. The overexpressed ROCK2 plays an important role in the pathogenesis of drug-resistant epilepsy. ROCK2 accelerates astrocytes cell cycle progression via the activation of Stat3 pathway likely provides the key to explaining the process of epileptogenesis.

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Crenolanib is active against models of drug-resistant FLT3-ITD−positive acute myeloid leukemia

Blood ◽

10.1182/blood-2013-07-513044 ◽

2013 ◽

Vol 122 (22) ◽

pp. 3607-3615 ◽

Cited By ~ 114

Author(s):

Eric I. Zimmerman ◽

David C. Turner ◽

Jassada Buaboonnam ◽

Shuiying Hu ◽

Shelley Orwick ◽

...

Keyword(s):

Myeloid Leukemia ◽

Kinase Inhibitor ◽

Drug Resistant ◽

Activating Mutations ◽

Flt3 Inhibitor ◽

Key Points ◽

Acute Myeloid

Key Points The tyrosine kinase inhibitor crenolanib has type 1 inhibitor properties and has potent activity against FLT3-activating mutations. Crenolanib is active in vitro and in vivo against FLT3 inhibitor-resistant FLT3-ITD/D835 mutations.

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Potential Therapeutic Benefit of Combining Gefitinib and Tamoxifen for Treating Advanced Lung Adenocarcinoma

BioMed Research International ◽

10.1155/2015/642041 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Chien-Ming Liu ◽

Kuo-Liang Chiu ◽

Tzu-Sheng Chen ◽

Shang-Miao Chang ◽

Shu-Yun Yang ◽

...

Keyword(s):

Cytotoxic Effect ◽

Kinase Inhibitor ◽

Growth Factor Receptor ◽

In Vitro Study ◽

Therapeutic Benefit ◽

Egfr Mutations ◽

Driver Mutations ◽

Therapeutic Benefits ◽

Potential Therapeutic Benefit

Introduction. Epidermal growth factor receptor (EGFR) mutations are known as oncogene driver mutations and with EGFR mutations exhibit good response to the EGFR tyrosine kinase inhibitor Gefitinib. Some studies have shown that activation of estrogen and estrogen receptorαorβ(ERα/β) promote adenocarcinoma. We evaluated the relationship between the two receptors and the potential therapeutic benefit with Gefitinib and Tamoxifen.Methods. We assessed the association between EGFR mutations as well as ERα/βexpression/location and overall survival in a cohort of 55 patients with LAC from a single hospital. PC9 (EGFR exon 19 deletion mutant; Gefitinib-vulnerable cells) and A549 (EGFR wild type; Gefitinib-resistant cells) cancer cells were used to evaluate the in vitro therapeutic benefits of combining Gefitinib and Tamoxifen.Results. We found that the cytosolic but not the nuclear expression of ERβwas associated with better OS in LAC tumors but not associated with EGFR mutation. The in vitro study showed that combined Gefitinib and Tamoxifen resulted in increased apoptosis and cytosolic expression of ERβ. In addition, combining both medications resulted in reduced cell growth and increased the cytotoxic effect of Gefitinib.Conclusion. Tamoxifen enhanced advanced LAC cytotoxic effect induced by Gefitinib by arresting ERβin cytosol.

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Observations of Frozen-Hydrated Microtubules

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100181270 ◽

1990 ◽

Vol 48 (1) ◽

pp. 504-505

Author(s):

D. Chrétien ◽

D. Job ◽

R.H. Wade

Keyword(s):

Fourier Transforms ◽

Structural Complexity ◽

Image Contrast ◽

Phase Behaviour ◽

Experimental Conditions ◽

Thin Sections ◽

Surface Lattice ◽

Cilia And Flagella ◽

Outstanding Question

Microtubules are filamentary structures found in the cytoplasm of eukaryotic cells, where, together with actin and intermediate filaments, they form the components of the cytoskeleton. They have many functions and show various levels of structural complexity as witnessed by the singlet, doublet and triplet structures involved in the architecture of centrioles, basal bodies, cilia and flagella. The accepted microtubule model consists of a 25 nm diameter hollow tube with a wall made up of 13 paraxial protofilaments (pf). Each pf is a string of aligned tubulin dimers. Some results have suggested that the pfs follow a superhelix. To understand how microtubules function in the cell an accurate model of the surface lattice is one of the requirements. For example the 9x2 architecture of the axoneme will depend on the organisation of its component microtubules. We should also note that microtubules with different numbers of pfs have been observed in thin sections of cellular and of in-vitro material. An outstanding question is how does the surface lattice adjust to these different pf numbers?We have been using cryo-electron microscopy of frozen-hydrated samples to study in-vitro assembled microtubules. The experimental conditions are described in detail in this reference. The results obtained in conjunction with thin sections of similar specimens and with axoneme outer doublet fragments have already allowed us to characterise the image contrast of 13, 14 and 15 pf microtubules on the basis of the measured image widths, of the the image contrast symmetry and of the amplitude and phase behaviour along the equator in the computed Fourier transforms. The contrast variations along individual microtubule images can be interpreted in terms of the geometry of the microtubule surface lattice. We can extend these results and make some reasonable predictions about the probable surface lattices in the case of other pf numbers, see Table 1. Figure 1 shows observed images with which these predictions can be compared.

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Superoxide, Xanthine Oxidase and Platelet Reactions: Further Studies on Mechanisms by which Oxidants Influence Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650190 ◽

1981 ◽

Vol 45 (03) ◽

pp. 290-293 ◽

Cited By ~ 9

Author(s):

Peter H Levine ◽

Danielle G Sladdin ◽

Norman I Krinsky

Keyword(s):

Superoxide Dismutase ◽

Cytochrome C ◽

Xanthine Oxidase ◽

Direct Effect ◽

Platelet Function ◽

Experimental Conditions ◽

Release Reaction

SummaryIn the course of studying the effects on platelets of the oxidant species superoxide (O- 2), Of was generated by the interaction of xanthine oxidase plus xanthine. Surprisingly, gel-filtered platelets, when exposed to xanthine oxidase in the absence of xanthine substrate, were found to generate superoxide (O- 2), as determined by the reduction of added cytochrome c and by the inhibition of this reduction in the presence of superoxide dismutase.In addition to generating Of, the xanthine oxidase-treated platelets display both aggregation and evidence of the release reaction. This xanthine oxidase induced aggreagtion is not inhibited by the addition of either superoxide dismutase or cytochrome c, suggesting that it is due to either a further metabolite of O- 2, or that O- 2 itself exerts no important direct effect on platelet function under these experimental conditions. The ability of Of to modulate platelet reactions in vivo or in vitro remains in doubt, and xanthine oxidase is an unsuitable source of O- 2 in platelet studies because of its own effects on platelets.

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