Increased Tyrosine Kinase Activity Research Articles

Global DNA Methylation analysis of imatinib resistant and sensitive K562 cells

OBJECTIVE: Chronic myeloid leukemia (CML) is a hematological disease which is known by the presence of Philadelphia chromosome (Ph+). BCR-ABL protein is expressed by Ph+ chromosome, represents constant increased tyrosine kinase activity. Imatinib (IMA) is a tyrosine kinase inhibitor (TKI) which is utilized as a first line treatment in CML. Emergence of IMA resistance at some point of therapy leads to treatment failure. DNA methylation is considered to be the most stable epigenetic change and several studies have shown that epigenetic changes may play a role in drug resistance. We investigated the global methylation profile of IMA-sensitive K562S, IMA-resistant K562R and IMA-resistant and adherent K562R (K562R-adh) cells to determine whether epigenetic reprogramming is involved in the resistance to IMA and the change in phenotype due to this resistance. MATERIAL AND METHODS: In this study, morphologically distinct, IMA-sensitive K562S and 5µM IMA-resistant K562R and K562R-adh in-vitro CML cell models were used to analyze the global DNA methylation profile. After DNA was isolated from the cells, global 5mC DNA methylation profiles were investigated by ELISA using equal amounts of DNA. RESULTS: Compared to K562S, the global methylation of K562R showed an increase in DNA methylation profile, but this increase in methylation was not statistically significant. Whereas, a slight hypermethylation was observed in the DNA of the K562R-adh vs K562S and K562R-adh vs K562R which is statistically significant. We observed slight hypermethylation in IMA-resistant cells lines versus to the IMA-sensitive cell line. CONCLUSION: Our observed differences in 5methyl-Cytosine on CpG islands (5mC) in K562S versus K562R and K562R-adh cell lines suggest that the DNA methylation alteration in resistant cells may partly contributed in phenotype switching.

The molecular signature of BCR::ABLP210 and BCR::ABLT315I in a Drosophila melanogaster chronic myeloid leukemia model

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder resulting from a balanced translocation leading to BCR::ABL1 oncogene with increased tyrosine kinase activity. Despite the advancements in the development of tyrosine kinase inhibitors (TKIs), the T315I gatekeeper point mutation in the BCR::ABL1 gene remains a challenge. We have previously reported in a Drosophila CML model an increased hemocyte count and disruption in sessile hemocyte patterns upon expression of BCR::ABL1p210 and BCR::ABL1T315I in the hemolymph. In this study, we performed RNA sequencing to determine if there is a distinct gene expression that distinguishes BCR::ABL1p210 and BCR::ABL1T315I. We identified six genes that were consistently upregulated in the fly CML model and validated in adult and pediatric CML patients and in a mouse cell line expressing BCR::ABL1T315I. This study provides a comprehensive analysis of gene signatures in BCR::ABL1p210 and BCR::ABL1T315I, laying the groundwork for targeted investigations into the role of these genes in CML pathogenesis.

Analyzing tyrosine kinase activity in head and neck cancer by functional kinomics: Identification of hyperactivated Src family kinases as prognostic markers and potential targets.

Signal transduction via protein kinases is of central importance in cancer biology and treatment. However, the clinical success of kinase inhibitors is often hampered by a lack of robust predictive biomarkers, which is also caused by the discrepancy between kinase expression and activity. Therefore, there is a need for functional tests to identify aberrantly activated kinases in individual patients. Here we present a systematic analysis of the tyrosine kinases in head and neck cancer using such a test-functional kinome profiling. We detected increased tyrosine kinase activity in tumors compared with their corresponding normal tissue. Moreover, we identified members of the family of Src kinases (Src family kinases [SFK]) to be aberrantly activated in the majority of the tumors, which was confirmed by additional methods. We could also show that SFK hyperphosphorylation is associated with poor prognosis, while inhibition of SFK impaired cell proliferation, especially in cells with hyperactive SFK. In summary, functional kinome profiling identified SFK to be frequently hyperactivated in head and neck squamous cell carcinoma. SFK may therefore be potential therapeutic targets. These results furthermore demonstrate how functional tests help to increase our understanding of cancer biology and support the expansion of precision oncology.

Evaluation of the neurotoxic effects of engineered nanomaterials in C57BL/6J mice in 28-day oral exposure studies

In recent years, concerns have emerged about the potential neurotoxic effects of engineered nanomaterials (NMs). Titanium dioxide and silver are among the most widely used types of metallic NMs. We have investigated the effects of these NMs on behaviour and neuropathology in male and female C57BL/6J mice following 28-day oral exposure with or without a 14-day post-exposure recovery. The mice were fed ad libitum with food pellets dosed with 10 mg/g TiO2, 2 mg/g polyvinylpyrrolidone-coated Ag or control pellets. Behaviour was evaluated by X-maze, open field, string suspension and rotarod tests. Histological alterations were analysed by immunohistochemistry and brain tissue homogenates were investigated for markers of oxidative stress, inflammation and blood-brain barrier disruption. Effects of the NMs on tyrosine and serine/threonine protein kinase activity in mouse brains were investigated by measuring kinase activity on peptide microarrays.Markers of inflammation, oxidative stress and blood-brain barrier integrity were not significantly affected in the male and female mice following exposure to Ag or TiO2. Both types of NMs also revealed no consistent significant treatment-related effects on anxiety and cognition. However, in the Ag NM exposed mice altered motor performance effects were observed by the rotarod test that differed between sexes. At 1-week post-exposure, a diminished performance in this test was observed exclusively in the female animals. Cortex tissues of female mice also showed a pronounced increase in tyrosine kinase activity following 28 days oral exposure to Ag NM. A subsequent Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) based toxicokinetic study in female mice revealed a rapid and persistent accumulation of Ag in various internal organs including liver, kidney, spleen and the brain up to 4 weeks post-exposure. In conclusion, our study demonstrated that subacute exposure to foodborne TiO2 and Ag NMs does not cause substantial neuropathological changes in mice. However, the toxicokinetic and specific toxicodynamic findings indicate that long-term exposures to Ag NM can cause neurotoxicity, possibly in a sex-dependent manner.

Pathogenic variants causing ABL1 malformation syndrome cluster in a myristoyl-binding pocket and increase tyrosine kinase activity

ABL1 is a proto-oncogene encoding a nonreceptor tyrosine kinase, best known in the somatic BCR-ABL fusion gene associated with chronic myeloid leukaemia. Recently, germline missense variants in ABL1 have been found to cause an autosomal dominant developmental syndrome with congenital heart disease, skeletal malformations and characteristic facies. Here, we describe a series of six new unrelated individuals with heterozygous missense variants in ABL1 (including four novel variants) identified via whole exome sequencing. All the affected individuals in this series recapitulate the phenotype of the ABL1 developmental syndrome and additionally we affirm that hearing impairment is a common feature of the condition. Four of the variants cluster in the myristoyl-binding pocket of ABL1, a region critical for auto-inhibitory regulation of the kinase domain. Bio-informatic analysis of transcript-wide conservation and germline/somatic variation reveals that this pocket region is subject to high missense constraint and evolutionary conservation. Functional work to investigate ABL1 kinase activity in vitro by transient transfection of HEK293T cells with variant ABL1 plasmid constructs revealed increased phosphorylation of ABL1-specific substrates compared to wild-type. The increased tyrosine kinase activity was suppressed by imatinib treatment. This case series of six new patients with germline heterozygous ABL1 missense variants further delineates the phenotypic spectrum of this condition and recognises microcephaly as a common finding. Our analysis supports an ABL1 gain-of-function mechanism due to loss of auto-inhibition, and demonstrates the potential for pharmacological inhibition using imatinib.

Follow-up form for adult patients with leukemia using Imatinib

Introduction: Chronic Myeloid Leukemia is a type of malignant blood neoplasia that is mainly characterized by the presence of the Philadelphia chromosome that originates the oncoprotein BCR-ABL. It has increased tyrosine kinase activity, thus causing changes in intracellular signaling pathways and promoting uncontrolled proliferation, cellular dysfunction and absence of apoptosis. Despite the low incidence of the disease, 1.5 cases per 100,000 inhabitants, therapy with Imatinib, a potent inhibitor of BCR-ABL, changed the prognosis of the disease and extended the life expectancy of patients, transforming a fatal disease into a chronic condition. Objective: To develop a form for regular monitoring of these patients by the clinical pharmacist to ensure safe and effective pharmacotherapy. Methodology: Narrative-type literature review was initiated from the problem question: ‘“What data should be collected by the Clinical Pharmacist during pharmaceutical assistance to assess and improve adherence in adult patients with Chronic Myeloid Leukemia using Imatinib?” , followed by search for articles in PubMed databases and government websites. Subsequently, work selection and critical analysis were carried out to build a model form to be applied in the act of dispensing. Result and Discussion. Eleven articles were selected. The patient’s low adherence to the treatment of chronic myeloid leukemia can result in potentially life-threatening. Therefore, the follow-up form to be applied by the clinical pharmacist when dispensing imatinib mesylate is a viable and low-cost strategy to improve adherence to pharmacotherapy and promote a better response to treatment. Conclusion: The systematic and standardized documentation of adherence and adverse effects by the Clinical pharmacist allows the implementation of actions by the multidisciplinary team of continuous improvements in order to maximize the quality of care provided to patients as well as the quality of life.

PLCγ1‑dependent invasion and migration of cells expressing NSCLC‑associated EGFR mutants.

The increased tyrosine kinase activity of non‑small cell lung cancer (NSCLC)‑associated epidermal growth factor receptor (EGFR) mutants results in deregulated pathways that contribute to malignant cell survival, tumor progression and metastasis. Previous studies investigating lung cancer‑associated EGFR have focused on the prognostic implications of receptor kinase mutations in patients with NSCLC; however, the role of EGFR mutations in tumor cell invasion and migration remains undetermined. The present study was designed to investigate the role of NSCLC‑associated mutant EGFR‑driven signaling pathways in cell proliferation and invasion. Non‑endogenous EGFR‑expressing 293cells stably expressing EGFR mutants that are sensitive or resistant to Food and Drug Administration(FDA)‑approved EGFR‑targeted tyrosine kinase inhibitors(TKIs) were used in the present study. The experiments demonstrated an increased phosphorylation of phospholipase (PLC)γ1, c‑Cbl, signal transducer and activator of transcription(Stat), extracellular regulated kinase (Erk)1/2, Akt, Shc and Gab1 proteins in cells expressing a mutant form, rather than the wild‑type receptor. As PLCγ1 is a known regulator of metastatic development, mutant receptor‑mediated PLCγ1 activation was further evaluated. To examine the effects of EGFR and PLCγ1 phosphorylation, the metastatic potential of cells expressing mutants was investigated using wound healing, Transwell cell migration and invasion assays. The inhibition of receptor phosphorylation with the 1st, 2nd and 3rdgeneration TKIs, gefitinib, afatinib, osimertinib, respectively, reduced PLCγ1 phosphorylation, and reduced the invasive and migratory potential of 293cells, confirming PLCγ1 as one of the probable downstream effectors of mutant EGFR signaling. However, the PLC inhibitor, U73122, inhibited cell migration and invasion without affecting EGFR signaling and PLCγ1 phosphorylation. Notably, U73122 reduced Akt and Erk1/2 phosphorylation within 25min of its application; however, 100%cell viability was recorded even after 48h. Upon further investigation, proliferative signaling pathways remained active at 48h, in accordance with cell viability. Therefore, the present study concludes that mutant receptor‑mediated PLCγ1 activation may play a significant role in the migration and invasion of NSCLC tumors; however, its regulatory role in tumor cell proliferation warrants further investigation and validation in lung tumor cell lines harboring EGFR mutations.

Характеристика мутацій кіназного домену гена BCR/ABL1 у пацієнтів з хронічною мієлоїдною лейкемією, первинно резистентних до терапії іматинібом

The chronic myeloid leukemia (CML) development is associated with the formation of the BCR/ABL1 fusion gene and the BCR/ABL1 protein with increased tyrosine kinase activity. Despite the high efficiency of targeted therapy, up to 30% of patients do not respond on such therapy i.e. are primary resistant. The presence of BCR/ABL1 kinase domain mutations is considered to be one of the reasons of tyrosin kinase inhibitors resistance. To evaluate the frequency of BCR/ABL1 kinase domain mutations in Ukrainian cohort of CML patients with primary resistance to imatinib therapy, we retrospectively studied BCR/ABL1 kinase domain mutations in peripheral blood of 107 CML patients. The nucleotide sequence was determined by direct sequencing by Sanger. Mutations were reported in 45 of 107 (41.7%) CML patients. Two mutations at a time were revealed in 8 patients. So a total of 53 mutations were found out. Among them 49 were missense-mutations and 4 - deletions of different regions of the BCR/ABL1 kinase domain gene. The missense-mutations F359I/V (12 patients), T315I (8 patients) and G250E (6 patients) were most common. By localization, the mutations majority (23 of 53) was in the P-loop, 10 mutations - in the contact site, 13 mutations - in the catalytic domain and 6 – in the A-loop. Of the detected mutations, 26 (49%) resulted in a disruption of the hydrogen bond between BCR/ABL1-tyrosine kinase and imatinib. Significant reduction in overall survival was found in patients with BCR/ABL1 kinase domain mutations compared with patients with wild-type of BCR/ABL1 gene (p=0.018). The estimated 3-year overall survival was 83.4% (95% CI: 77.0%-89.8%) and 94.3% (95% CI: 91.0%-97.3%), respectively. Therefore, mutations of the BCR/ABL1 kinase domain are one of the mechanisms of primary resistance in CML patients on imatinib therapy. The occurrence of BCR/ABL1 gene mutations impairs the prognosis of imatinib therapy response.

Functional Analysis of Somatic Mutations Affecting Receptor Tyrosine Kinase Family in Metastatic Colorectal Cancer.

Besides the detection of somatic receptor tyrosine kinases (RTK) mutations in tumor samples, the current challenge is to interpret their biological relevance to give patients effective targeted treatment. By high-throughput sequencing of the 58 RTK exons of healthy tissues, colorectal tumors, and hepatic metastases from 30 patients, 38 different somatic mutations in RTKs were identified. The mutations in the kinase domains and present in both tumors and metastases were reconstituted to perform an unbiased functional study. Among eight variants found in seven RTKs (EPHA4-Met726Ile, EPHB2-Val621Ile, ERBB4-Thr731Met, FGFR4-Ala585Thr, VEGFR3-Leu1014Phe, KIT-Pro875Leu, TRKB-Leu584Val, and NTRK2-Lys618Thr), none displayed significantly increased tyrosine kinase activity. Consistently, none of them induced transformation of NIH3T3 fibroblasts. On the contrary, two RTK variants (FGFR4-Ala585Thr and FLT4-Leu1014Phe) caused drastic inhibition of their kinase activity. These findings indicate that these RTK variants are not suitable targets and highlight the importance of functional studies to validate RTK mutations as potential therapeutic targets.

CircBA9.3 supports the survival of leukaemic cells by up-regulating c-ABL1 or BCR-ABL1 protein levels

The unchecked tyrosine kinase activity of BCR-ABL1 contributes to the immortality of leukaemic cells. Therefore, this oncogene is a highly important therapeutic target for chronic myelogenous leukaemia (CML). Tyrosine kinase inhibitors (TKIs) are an excellent drug treatment for CML patients. However, there are still some patients who are not responsive to TKIs. We found that a novel circular RNA (circRNA), named circBA9.3, is derived from BCR-ABL1. CircBA9.3 can efficiently promote the proliferation and inhibit apoptosis of cancer cells. In addition, some patients with TKI resistance have elevated circBA9.3 expression, which is positively correlated with the level of BCR-ABL1. Furthermore, circBA9.3 is predominantly located in the cytoplasm and enhances c-ABL1 and BCR-ABL1 oncoprotein expression. Thus, circBA9.3 is a molecule associated with increased tyrosine kinase activity that promotes resistance against TKI therapy. In this study, we provided a new potential target for the treatment of TKI-resistant CML patients.

Regulation of dual specificity phosphatases in breast cancer during initial treatment with Herceptin: a Boolean model analysis

Background25% of breast cancer patients suffer from aggressive HER2-positive tumours that are characterised by overexpression of the HER2 protein or by its increased tyrosine kinase activity. Herceptin is a major drug used to treat HER2 positive breast cancer. Understanding the molecular events that occur when breast cancer cells are exposed to Herceptin is therefore of significant importance. Dual specificity phosphatases (DUSPs) are central regulators of cell signalling that function downstream of HER2, but their role in the cellular response to Herceptin is mostly unknown. This study aims to model the initial effects of Herceptin exposure on DUSPs in HER2-positive breast cancer cells using Boolean modelling.ResultsWe experimentally measured expression time courses of 21 different DUSPs between 0 and 24 h following Herceptin treatment of human MDA-MB-453 HER2-positive breast cancer cells. We clustered these time courses into patterns of similar dynamics over time. In parallel, we built a series of Boolean models representing the known regulatory mechanisms of DUSPs and then demonstrated that the dynamics predicted by the models is in agreement with the experimental data. Furthermore, we used the models to predict regulatory mechanisms of DUSPs, where these mechanisms were partially known.ConclusionsBoolean modelling is a powerful technique to investigate and understand signalling pathways. We obtained an understanding of different regulatory pathways in breast cancer and new insights on how these signalling pathways are activated. This method can be generalized to other drugs and longer time courses to better understand how resistance to drugs develops in cancer cells over time.

Abstract 3163: Discovery of a kinome signature predicting sensitivity and resistance to RAF-MAPK pathway inhibitors in melanoma

Abstract Introduction RAF-MAPK pathway inhibition with BRAF inhibitors or the combination of BRAF and MEK inhibitors has become the mainstay of therapy of metastatic melanoma harboring BRAF V600 mutations with a response rate as high as 50-75 %. A minority of patients present with primary resistance while all patients develop secondary resistance. We explored a reversed translational approach using a novel multiplex kinase assay to first identify individuals not responding to therapy using baseline biopsy samples and second to understand the mechanism of resistance using patient-derived cell lines. Method Pre-dose tissues from 4 non-responders and 3 responders to vemurafenib monotherapy, all harboring the V600E mutation, were profiled for Serine-Threonine Kinase (STK) activities on a PamChip® peptide microarray in the presence and absence of dabrafenib. For molecular studies three melanoma cell lines, all with BRAF V600E mutations, were made resistant to vemurafenib. Protein Tyrosine Kinase (PTK) and STK activities in lysates of sensitive or resistant cells were measured +/- dabrafenib, trametinib or both. Lysates were blotted against a selected panel of (phospho)proteins. Results In tissue lysates, concentration-dependent ex vivo inhibition with dabrafenib was stronger in the patients that were clinical responders than in non-responders. This difference in inhibition of STK activity by dabrafenib was confirmed in the cell lines that were made resistant against vemurafenib. IC50s for the sensitive cell lines were below 0.5 µM and varied from 10-21 µM for cell lines with acquired resistance. PTK activity was increased in resistant cell lines. STK activity was lower, but large differences exist among the cell lines. Common features of resistance were increased activity of receptor tyrosine kinases, Src family kinases and AKT signaling. These results were confirmed by Western blot analysis. Dabrafenib (10 µM) caused inhibition of STK activity while trametinib (10 µM) gave some activation. Dabrafenib (0.5 µM) gave strong inhibition of PTK activity, both in tissue and cell lines. Trametinib had hardly any inhibitory effect. Interestingly, the combination of dabrafenib and trametinib had an antagonistic effect on the STK activity and a synergistic effect on PTK activity, resulting in a stronger inhibition of kinase activity. Conclusion Resistance of tumors and cell lines to vemurafenib can affect kinase activity profiles as detected pre-dose using a multiplex kinase assay. Dabrafenib inhibition of PTK and STK activity is stronger in sensitive cell lines. The overall activation of kinases in a lysate upon MEK inhibition suggests MEK as suppressor of kinase activity. Resistance to MAPK pathway inhibitors could be linked to an increase in tyrosine kinase activity and AKT pathway activity. Biomarkers are needed to identify patients in need of combined MAPK pathway inhibition. Citation Format: Riet Hilhorst, Mohammed Krayem, Adrienne van den Berg, Fabrice Journe, Liesbeth Hovestad-Bijl, Tim van den Hooven, Rik de Wijn, Philippe Aftimos, Ghanem Ghanem, Rob Ruijtenbeek. Discovery of a kinome signature predicting sensitivity and resistance to RAF-MAPK pathway inhibitors in melanoma [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 3163. doi:10.1158/1538-7445.AM2017-3163

Oxidative damage and cell signaling transduction in patients of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) was the first human malignancy shown to be consistently associated with a chromosomal abnormality, the Philadelphia chromosome. At the gene level, the Philadelphia chromosome is the result of breaks on chromosomes 9 and 22 with a reciprocal translocation of the distal genetic material. This translocation forms the new hybrid BCR-ABL oncogene, an abnormal 8.5-kb RNA that encodes a 210-kDa fusion protein, which, presumably through its increased tyrosine kinase activity, changes normal hematopoietic cells into CML cells [1]. Oxidative stress is a biochemical condition that occurs when intracellular antioxidants are unable to neutralize pro-oxidants such as reactive oxidant species (ROS). These ROS damage membranes, DNA, lipids, proteins, and carbohydrates, eventually causing cell injury and death. ROS contribute to several cellular functions, including the regulation of signal transduction, gene expression, and cell proliferation [2]. Nuclear factor kappa B (NF-κB) is a redox-sensitive transcription factor that is mostly cytoplasmic and maintained in the cytoplasm by interaction with its inhibitor IκB-α. NF-κB pathway plays an important role in the control of cell proliferation, differentiation, apoptosis, stress response, cell signaling transduction, and other physiological processes [3]. ROS can activate NF-κB signal transduction pathways, which in turn lead to the transcription of genes involved in cell growth regulatory pathways. A number of somatic mutations have been identified in cancer, like transversion mutations of p53. These key genes have been linked to cancer progression, and the mutations found in them can be produced by ROS. Although the direct link between ROS modification of DNA and mutations of these genes remains to be established, they should be considered important candidates for the induced carcinogenesis because mutations in these genes could be responsible for tumor initiation as well as progression. ROS have also been shown to increase the production of the angiogenic factor such as vascular endothelial growth factor (VEGF) in tumor cells resulting in carcinogenesis. In an attempt to explore the possibility that cellular redox status may up-regulate NF-κB signaling, the present study was designed to correlate the alterations in the pro-oxidant/antioxidant status with the up-regulation of the nuclear transcription factor NF-κB, pro-inflammatory cytokine VEGF, and tumor suppressor gene p53 in the development/progression of CML. Case control study consisted of 40 clinically diagnosed CML patients from the Department of Medicine, University Hospital, Banaras Hindu University (Varanasi, India), with cytogenetic analysis to confirm the presence of Philadelphia (Ph) chromosome. Exclusion criteria for enrolment in the study group were patients with clinical or pathologic evidence of cancer at any other site or having received any type of neo-adjuvant therapy along with the presence of liver dysfunction, diabetes mellitus, heart failure, or renal failure. The mean age of the CML patients was 36.7 ± 11.9 years. The mean leukocyte count was 80,410 per mm. Themean spleen size of patients measured by ultrasonography was 21.1 ± 3.5 cm, and was found to be significantly larger than controls. The Philadelphia chromosomewas present in all of 40 patients with CML, which confirmed the BCR/ABL translocation. Forty age and sex-matched healthy volunteers having socioeconomic status similar to that of patients served as controls. None of the patients and controls was under antioxidant supplementation. The venous blood was drawn from patients and healthy volunteers in sterile tubes for various biochemical and hematologic investigations. The study protocol was approved by the ethical committee of the Institute of Medical Sciences, Banaras Hindu University. Informed consent of each patient and healthy volunteers was obtained purely for research purpose. The oxidative stress parameters 8-hydroxydeoxyguanosine (8-OHdG), protein carbonyl (PC), and total antioxidant status (TAS) were measured [4–6]. NF-κB p65 subunit DNA-binding activity, VEGF, and p53 levels were estimated to correlate the up-regulation of nuclear transcription factor/cytokine level with increased oxidative stress so as to determine the role of oxidative microenvironment in the progression of disease. All the data were expressed as the mean ± standard error of the mean (SEM). Data were analyzed statistically by independent Student’s t-test for comparison of parametric variables. Either linear or nonlinear regression analysis was applied for association studies. All statistical analyses were two-tailed and a value of Acta Biochim Biophys Sin, 2015, 47(6), 474–476 doi: 10.1093/abbs/gmv029 Advance Access Publication Date: 28 April 2015 New Phenomenon

152P - Epidermal Growth Factor Receptor Signaling Conferred Acquired Crizotinib Resistance to a Non-Small Cell Lung Cancer Cell Line Harboring the Slc34A2-Ros1 Fusion Gene

ABSTRACT Aim: The ROS1 inhibitor crizotinib produces a remarkable response in patients with non-small cell lung cancer (NSCLC) harboring the ROS1 fusion gene (J Clin Oncol 2013 abstr 8032); however, acquired crizotinib resistance is inevitable, but the mechanism is unclear. Methods: HCC78 cells harboring the SLC34A2-ROS1 gene alteration were used to model acquired crizotinib resistance to explore alternative molecular mechanisms. The resistant cell line (HCC78R) was established from HCC78 cells after continuous exposure to crizotinib for 4 months in vitro. Results: The 50% inhibitory concentration (IC50) of crizotinib in HCC78R cells (4085.9 nM) was 47-fold larger than the IC50 in parental HCC78 cells (85.8 nM) (p = 0.0145). The expression of ROS1 protein was decreased significantly in HCC78R cells, as compared to HCC78 cells. Crizotinib inhibited the phosphorylation of AKT or ERK and downstream ROS1 signaling in HCC78 cells, but did not inhibit this in HCC78R. A receptor tyrosine kinas array showed that there was no increased tyrosine kinase activation in HCC78R cells, as compared to HCC78 cells. Interestingly, however, the phosphorylation of EGFR was maintained under 2,000 nM crizotinib exposure in HCC78R cells, but inhibited in parental HCC78 cells. Therefore, we hypothesize that the EGFR signaling pathway plays a role in HCC78R cells. Expectedly, HCC78R cells were very sensitive to EGFR-TKI in a cell proliferation assay (IC50: 117 nM vs. > 10,000 nM for HCC78R vs. HCC78 cells). Consistently, the addition of EGF to the culture medium rendered the parental HCC78 cells resistant to crizotinib. No activation of EGFR mutations were found in HCC78R cells. Finally, foretinib, a multi-kinase inhibitor of ROS1 and EGFR, effectively inhibited the proliferation of both HCC78R and HCC78 cells. Conclusions: These studies suggest that EGFR signaling plays a role in crizotinib resistance in lung cancer cells harboring ROS1 fusion genes. Disclosure: All authors have declared no conflicts of interest.

Premature lethality, hyperactivity, and aberrant phosphorylation in transgenic mice expressing a constitutively active form of Fyn

The kinase Fyn, the microtubule-associated protein tau and the peptide amyloid-β (Aβ) constitute a toxic triad in Alzheimer's disease (AD). Tau's subcellular localization is mainly regulated by phosphorylation whereas Fyn's localization is dictated by palmitoylation targeting it to the plasma membrane in a reversible manner. We have previously shown that tau is required for Fyn to be targeted to the dendritic spine. We had also shown that a truncated form of tau (Δtau) that accumulates in the cell soma is capable of trapping Fyn and preventing it from entering the spine. Here we determined that palmitoylation is required for Fyn's membrane and spine localization. We further evaluated the functional consequences of neuronal over-expression of the constitutively active Y531F mutant form of Fyn (FynCA) in transgenic mice. We found that the FynCA transgenic mice displayed a reduced weight, a massively reduced lifespan and a high level of hyperactivity. The lifespan of the FynCA mice was only slightly extended by crossing them with Δtau transgenic mice, possibly reflecting differences in expression patterns of the transgenes and high levels of transgenic FynCA compared to endogenous Fyn. Analysis of synaptosomes revealed that FynCA accumulated at high levels in the spine, resulting in increased levels of the NMDA receptor subunit NR2b phosphorylated at residue Y1472. Tau was strongly phosphorylated at the AT8 epitope S202/T205 as shown by Western blot and immunohistochemistry indicating that an increased tyrosine kinase activity of Fyn has down-stream consequences for serine/threonine-directed phosphorylation.

The role of mitochondrial DNA damage and repair in the resistance of BCR/ABL-expressing cells to tyrosine kinase inhibitors.

Chronic myeloid leukemia (CML) is a hematological malignancy that arises from the transformation of stem hematopoietic cells by the fusion oncogene BCR/ABL and subsequent clonal expansion of BCR/ABL-positive progenitor leukemic cells. The BCR/ABL protein displays a constitutively increased tyrosine kinase activity that alters many regulatory pathways, leading to uncontrolled growth, impaired differentiation and increased resistance to apoptosis featured by leukemic cells. Current CML therapy is based on tyrosine kinase inhibitors (TKIs), primarily imatinib, which induce apoptosis in leukemic cells. However, some patients show primary resistance to TKIs while others develop it in the course of therapy. In both cases, resistance may be underlined by perturbations in apoptotic signaling in leukemic cells. As mitochondria may play an important role in such signaling, alteration in mitochondrial metabolism may change resistance to pro-apoptotic action of TKIs in BCR/ABL-positive cells. Because BCR/ABL may induce reactive oxygen species and unfaithful DNA repair, it may affect the stability of mitochondrial DNA, influencing mitochondrial apoptotic signaling and in this way change the sensitivity of CML cells to TKIs. Moreover, cancer cells, including BCR/ABL-positive cells, show an increased level of glucose metabolism, resulting from the shift from oxidative phosphorylation to glycolysis to supply ATP for extensive proliferation. Enhanced level of glycolysis may be associated with TKI resistance and requires change in the expression of several genes regulated mostly by hypoxia-inducible factor-1α, HIF-1α. Such regulation may be associated with the impaired mitochondrial respiratory system in CML cells. In summary, mitochondria and mitochondria-associated molecules and pathways may be attractive targets to overcome TKI resistance in CML.

Response to Frontline Therapy with Second Generation Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia: Analysis of Outcome for b3a2 Vs. b2a2 Fusion Transcripts

AbstractAbstract 3781 Background:CML is characterized by the reciprocal translocation between chromosomes 9 and 22 [t(9;22)(q34;q11.2)], also known as the Philadelphia chromosome. This results in the formation of the BCR-ABL fusion gene, which is translated to a protein with increased tyrosine kinase activity. The breakpoint in the BCR gene can occur in different sites, most commonly between exons 13 and 15 which fuse to exon 2 of the ABL gene creating the b2a2 and b3a2 junctions. Previous studies have reported a better outcome for patients (pts) having a b2a2 transcript when treated with imatinib. To our knowledge there are no published reports analyzing the outcome of pts treated with a 2ndgeneration TKI as initial therapy for CML according to the fusion transcript. We analyzed the significance of these variations among pts with CML in chronic phase (CP) enrolled in 2 parallel trials of dasatinib and nilotinib as initial therapy. Patients and Methods:A total of 204 pts with CML in CP, treated at our institution between 2005 and 2012 with nilotinib or dasatinib as frontline therapy were included in this analysis. Among them 88 (43%) had the b2a2 transcript (median age of 47 years; range 18–80) at the start of therapy, 74 (36%) showed the b3a2 variant (median age of 47; range 17–82 years), and 42 (21%) presented both (median age 53; range 27–81). One patient had a b3a3 transcript and 2 pts showed an e1a2 transcript. Forty-two (48%) of those with b2a2 were treated with dasatinib and 46 (52%) with nilotinib; of the pts with b3a2 34 (46%) were treated with dasatinib and 40 (54%) with nilotinib, in the group of pts with both transcripts 24 (57%) received dasatinib 24, and 18 (43%) of them nilotinib (Table 1). Results:The Sokal risk group for those with the b2a2 transcript was high in 10%, intermediate in 29%, and low in 61%. For pts with b3a2 the risk classification was 5%, 25, and 70%, respectively. The group with both transcripts had 10%, 31% and 69%, respectively. Significant difference was observed in the time of molecular response and overall outcome between pts with b2a2 and those with b3a2 (CCyR 91% vs. 96%; MMR 75% vs. 91%, respectively). The 3-year probability of overall survival (OS) was 100% for both groups. The 3-year probability of event-free survival (EFS) was 93% and 99%, and the transformation-free survival (TFS) 99% and 98% for the b2a2 and the b3a2 group respectively (Table 2). On the 12 mo. follow-up one of the pts with e1a2 transcript achieved CCyR (treated with nilotinib), and the second patient (pt) major cytogenetic response (receiving dasatinib). The pt presenting with b3a3 was treated with dasatinib and achieved MMR by 3 mo. and CCyR at the 12 months follow-up. Conclusions:Although all pts whether they express b3a2 or b2a2 at diagnosis, have an excellent overall survival, those with b3a2 have a significantly higher rate of molecular responses and a trend for better EFS. These results mirror what we have previously reported for pts treated with imatinib as initial therapy. The biologic characteristics for this difference warrant further investigation.Table 1Treatment distribution in analyzed groupsTranscriptDasatinibNilotinibb2a242 (48%)46 (52%)b3a234 (46%)40 (54%)b2a2 and b3a224 (57%)18 (43%)Table 2Results of patients with variant BCR-ABL transcripts treated with 2ndgeneration TKIsb2a2b3a2b2a2+b3a2Overall CCyR90.4%95.7%95.1%Overall MMR75%91.5%95%12 mo. CCyR93.8%100%100%12 mo. MR66.7%98.184.7%3 years O.S.100%100%94%3 years EFS93%99%98%3 years TFS99%98%100% Disclosures:Kantarjian:Novartis: Research Funding; Bristol Myers Squibb: Research Funding; Ariad: Research Funding; Pfizer: Research Funding. Jabbour:Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Cortes:Novartis: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding.

Effect of Metformin on Tyrosine Kinase Autophosphorylation of Erythrocyte Insulin Receptor from Women with Polycystic Ovary Syndrome

Objective To document the possible effect of tyrosine kinase autophosphorylation on erythrocyte insulin-receptor after 12 weeks of metformin administration from the women with polycystic ovary syndrome (PCOS). Methods Thirty non-obese (BMI 2 ) healthy women with normal reproductive cycles were evaluated by conventional criteria as the control, 30 non-obese women with PCOS were categorized as nob-PCOS group, 40 obese women with PCOS (BMI ⩾ 25 kg/m 2 ) were categorized as ob-PCOS group. Subjects with PCOS were given metformin 850 mg/d for 12 weeks. The autophosphorylation of insulin receptor pY1158, pY1162/1163 and insulin receptor-β subunit (IR-β) from the solubilised erythrocyte were detected by ELISA. Results 1) Only the autophosphorylation level of pY1158 in nob-PCOS and ob-PCOS groups was lower (P 0.05), even insulin-stimulated insulin receptor (P>0.05) from both of PCOS groups. 2) After metformin administration, the pY1158, pY1162/pY1163 autophosphorylation levels were increased (P 0.05). In summary, oral administration of metformin led to an significant increase in tyrosine kinase activity in both groups of PCOS. Conclusion The impairment of tyrosine autophosphorylation at pY1158 may contribute to insulin resistance, the tyrosine kinase activity per receptor of solubilised erythrocytes were significantly increased by metformin administration and the effect of metformin on insulin-receptor tyrosine kinase activity appeared to be independent of either of these variables.

Evidence for BCR‐ABL‐dependent dysfunctions of iNKT cells from chronic myeloid leukemia patients

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem-cell malignancy characterized by the presence of the chimeric BCR-ABL oncoprotein with deregulated tyrosine-kinase (TK) activity. Although conventional T cells are acknowledged as important players in the control of CML, a possible modification of invariant NKT (iNKT) cells, known for their antitumoral activity, has not been established as yet. Here, we showed that the expression of perforin, CD95L, and promyelocytic leukemia zinc finger, a transcription factor required for maintenance of iNKT cell functions, was reduced or suppressed in CML patients at diagnosis, as compared with healthy individuals. The proliferation rate of blood iNKT cells in response to their cognate ligand was likewise diminished. These functional deficiencies were corrected in patients having achieved complete cytogenetic remission following TK inhibitor or IFN-α therapy. iNKT cells from CML patients in the chronic phase did not display increased TK activity, which argued against a direct autonomous action of BCR-ABL. Instead, we found that their anergic status originated from both intrinsic and APC-dependent dysfunctions. Our data demonstrate that chronic phase CML is associated with functional deficiencies of iNKT cells that are restored upon remission. These results suggest a possible contribution to disease control by TK inhibitor therapies.

Molecular and Structural Characterization of the SH3 Domain of AHI-1 in Regulation of Cellular Resistance of BCR-ABL+ Chronic Myeloid Leukemia Cells to Tyrosine Kinase Inhibitors

Abstract 966Chronic myeloid leukemia (CML) is a clonal multilineage myeloproliferative disorder characterized by the presence of the fusion gene BCR-ABL with increased tyrosine kinase activity. Imatinib mesylate (IM) and other BCR-ABL tyrosine kinase inhibitors (TKIs), including dasatinib (DA) and nilotinib (NL), have been introduced into clinical practice with remarkable effects on chronic phase CML. However, early relapses, acquired drug resistance, and persistence of leukemic stem cells remain problematic. Improved treatment approaches to target other key molecular elements active in CML stem/progenitor cells are needed. One candidate is AHI-1 (Abelson helper integration site 1), an oncogene that is highly deregulated in CML leukemic stem cells. It harbors two key domains, SH3 and WD40-repeat, which are known important mediators of protein-protein interactions. We recently demonstrated that AHI-1 physically interacts with BCR-ABL and JAK2 in CML cells and this interaction complex mediates transforming activity and TKI response/resistance of CML stem/progenitor cells. We have also shown that AHI-1 interacts independently with JAK2 and BCR-ABL via different binding sites to mediate their activities. In this study, we have characterized the biological and structural functions of the SH3 domain of AHI-1. To determine roles of the SH3 domain in regulation of cell proliferation and TKI response/resistance, several mutant forms, including SH3 domain deletion (SH3Δ), double WD40-repeat and SH3 domain deletion (SH3WD40Δ) and N-terminal deletion (N-terΔ, containing SH3 and WD40-repeat domains) were generated and stably transduced into BCR-ABL inducible BaF3 cells, in which the level of expression of BCR-ABL can be down-regulated by exposure to doxycycline. Overexpression of full-length Ahi-1 in BCR-ABL inducible cells resulted in fewer Annexin V+ apoptotic cells with doxycyclin (suppression of BCR-ABL) compared to BCR-ABL inducible cells (3 and 29% v.s.10 and 60% after 24 or 48 hours). Cells expressing the SH3Δ mutant and the SH3WD40D mutant displayed dramatically increased Annexin V+ cells (10, 77% and 34, 90% v.s.3 and 29%), while cells expressing the N-terΔ mutant had similar numbers of Annexin V+ cells compared to BCR-ABL inducible cells (6 and 41% v.s.10 and 60%). Similarly, BCR-ABL+ cells transduced with SH3Δ and SH3WD40D mutants displayed significantly increased apoptotic cells compared to cells transduced with full-length Ahi-1 in the presence of 2 μM IM (57, 87 vs. 26%), 2μM NL (65, 87 vs. 25%) and 150 nM DA (63, 96 vs. 34%) after 24 hour treatment. BCR-ABL+ cells transduced with the N-terδ mutant also showed more sensitivity to the drug treatments compared to the cells with the full-length Ahi-1(36% for IM, 40% for NL and 40% for DA), but with lower sensitivity than cells carrying the Ahi-1 SH3 domain deletion mutants, indicating that the SH3 domain of Ahi-1 plays a role in the mediation of TKI resistance. The crystal structure of the AHI-1 SH3 domain at 1.32-Å resolution revealed that the AHI-1SH3 domain adopts a canonical SH3 folding, but with an unusual C-terminal α helix. There are three large negatively charged patches, which are constructed by the n-Src loop, the end of the RT loop and the C-terminal helix, and this special feature may be involved in binding selectivity and specificity. PD1R peptide, known to interact with the PI3K SH3 domain, was used to model the binding pattern between AHI-1 SH3 domain and its ligands, and there may be formation of an “Arg-Arg-Trp” stack within the binding interface, which could be a targeting site for designing specific drugs. Moreover, using the AHI-1 SH3 domain as protein ‘bait’ in immunoprecipitation/mass spectrometry, Dynamin-2 was identified as a potential interacting partner of AHI-1; both AHI-1 and Dynamin-2 are involved in trafficking and signaling processes. In conclusion, the investigation of the structure of AHI-1 SH3 domain and its interacting proteins will thus provide invaluable insight in identification of key interaction sites in regulation of drug resistance and may be utilized for development of small molecule inhibitors for CML. Disclosures:No relevant conflicts of interest to declare.