Mon, Apr 2, 8:00 AM - 12:00 PM
1683/2 - ALK F1174L kinase activity as driver of cell proliferation in neuroblastoma cell
line models and neuroblastoma tumors Riet Hilhorst1, Liesbeth Hovestad1, Rik de
Wijn1, Dirk Pijnenburg1, Rob Ruijtenbeek1, Alexander Schramm2, Angelika Eggert2,
Johannes H. Schulte2. 1PamGene International BV, 's-Hertogenbosch, Netherlands;
2Dept. of Pediatric Hematology and Oncology, University Children's Hospital Essen,
Essen, Germany Poster Session
PO.CL01.04. Biomarkers for Personalized Cancer Therapy
Abstract
Number:
1683
Presentation
Title:
ALK F1174L kinase activity as driver of cell proliferation in
neuroblastoma cell line models and neuroblastoma tumors
Presentation
Time:
Monday, Apr 02, 2012, 8:00 AM -12:00 PM
Location:
McCormick Place West (Hall F), Poster Section 25
Poster
Section:
25
Poster Board
Number:
2
Author
Block:
Riet Hilhorst1, Liesbeth Hovestad1, Rik de Wijn1, Dirk Pijnenburg1, Rob
Ruijtenbeek1, Alexander Schramm2, Angelika Eggert2, Johannes H.
Schulte2. 1PamGene International BV, 's-Hertogenbosch, Netherlands;
2
Dept. of Pediatric Hematology and Oncology, University Children's
Hospital Essen, Essen, Germany
Abstract
Body:
Background.
Whereas ALK translocations play a role in tumor growth in selected
NSCLC cases, mutated forms of ALK have been shown to drive
development of neuroblastoma, the most common malignant extracranial
tumor of childhood. The mutation F1147L in the oncogene ALK
increases the kinase activity, causing deregulated proliferation of
sympatho-adrenal progenitor cells. We explored the use of kinase activity
measurements to identify ALK activity in cell lysates and patient derived
tissue lysates with the aim of identifying biomarkers to trace the activity
of ALK in tumors and the effect of ALK inhibitors on these marker
signals.
Methods.
Protein kinase activities were measured on PamChip® peptide
microarrays comprising 144 Tyr containing peptide sequences from
known human phosphorylation sites. The activity profiling experiments
involved recombinant ALK kinase, lysates of cell lines overexpressing
ALK and neuroblastoma tumors with elevated ALK F1174L presence.
Per microarray analysis 5 ug of total protein was used in the presence of
100 uM ATP. Experiments were performed in the presence and absence
of ALK kinase inhibitors crizotinib and NVP-TAE684. Real time kinetics
of 144 peptide phosphorylations were measured with a fluorescently
labeled antibody.
Results.
Phosphorylation profiles for neuroblastoma tumors, ALK overexpressing
cell lines and recALK were generated. For recALK 48 novel peptide
substrates were identified that showed signals depending on both ATP
and kinase concentration and inhibition by the ALK inhibitors. In ALK
overexpressing cell lines, but not in control cell lysates, signals on many
ALK substrate peptides were significantly elevated, and returned to
baseline level in the presence of ALK inhibitors. Whereas neuroblastomas
with low ALK expression gave similar kinase activity profiles, the tumors
harboring the ALK F1174L mutation were different, suggesting a more
complex regulation of phosphorylation activity in these tumors. All
tumors showed on-chip responses with ALK inhibitor drugs, but to
different extent, which could be a basis for response prediction biomarker
discovery.
Conclusion.
ALK activity of recombinant kinases, cell lysates and tumor lysates can
be detected on PamChip® peptide microarrays. Based on results with
model systems, this method shows promise to identify tumors that express
elevated ALK activity and to study response of this class to ALK
inhibitors.
Tue, Apr 3, 8:00 AM - 12:00 PM
3608/10 - Measurement of kinase activity in cancer cell lines and tumor tissue using a
tyrosine kinase peptide substrate array Mariette Labots, Kristy J. Gotink, Henk Dekker,
Johannes C. van der Mijn, Henk J. Broxterman, Maria N. Rovithi, Connie R. Jiménez,
Henk M.W. Verheul. VU University Medical Center, Amsterdam, Netherlands Poster
Session
PO.CL13.07. Biomarker Targets, Assays, and Models
Abstract
Number:
3608
Presentation
Title:
Measurement of kinase activity in cancer cell lines and tumor tissue using
a tyrosine kinase peptide substrate array
Presentation
Time:
Tuesday, Apr 03, 2012, 8:00 AM -12:00 PM
Location:
McCormick Place West (Hall F), Poster Section 23
Poster
Section:
23
Poster Board
Number:
10
Author
Block:
Mariette Labots, Kristy J. Gotink, Henk Dekker, Johannes C. van der
Mijn, Henk J. Broxterman, Maria N. Rovithi, Connie R. Jiménez, Henk
M.W. Verheul. VU University Medical Center, Amsterdam, Netherlands
Abstract
Body:
Introduction
Tyrosine kinases play an important role in tumor biology. Their activity
can be measured using a kinase peptide substrate array consisting of 144
Tyr residue-containing peptides (PamChip®, PamGene, Den Bosch, The
Netherlands). We evaluated this platform for the measurement of kinase
activity in tumor tissue and cancer cell lines under various experimental
conditions.
Methods
Lysates of colorectal and renal cancer cell lines, HCT116 and 786-0
respectively, were made using both Mammalian and Tissue Protein
Extraction Reagent (M-PER and T-PER, Thermo Scientific) and RadioImmunoprecipitation Assay (RIPA, home made) buffer. Lysates from
patient-derived tumor tissues were prepared by adding T-PER to several
10 μm cryoslides containing >50% tumor. After lysate incubation with
reaction buffer containing a fluorescent labeled antibody against phospotyrosine and ATP, kinase activity profiles were determined on kinetics of
recorded peptide substrate phosphorylation intensities. The effect of
protein and ATP concentration, different lysis buffers and number of
freeze-thaw cycles on basal kinase activity was studied. Sunitinib,
sorafenib and dasatinib, clinically available tyrosine kinase inhibitors
(TKIs), were used to differentially inhibit kinase activity in the lysates.
Results
Application of 2.5-15 µg protein in 40 µl sample mix per array revealed
linearly increasing phosphorylation signal intensities and initial velocities
(Vini) of the kinetic curve (R2 = 0.98). Increasing ATP concentrations
induced phosphorylation signal intensities, but above 400 µM the curve
deviated from linearity. Basal kinase activity profiles of cell lines and
tumor tissues were reproducible with CV’s below 15%, with good signalto-background ratios and low aspecific binding. Different lysis buffers
resulted in a maximum variation of phosphorylation signal intensity of
47±5.7% in both cell lines without affecting the actual profile. Quadruple
freeze-thawing of lysates did not affect signal intensities by more than
10%. Inhibition profiles of treated vs. control lysates were reproducible
within and between experiments, showing a higher and differential
number of inhibited peptides at increasing TKI concentrations. In contrast
to the ATP-independent inhibition of dasatinib, ATP-dependent inhibition
for sunitinib and sorafenib was demonstrated by combining a fixed drug
concentration with increasing concentrations of ATP up to 800 µM.
Conclusion
Kinase activity in lysates from cancer cell lines and patient-derived tumor
tissue can be reproducibly profiled with a tyrosine kinase peptide
substrate array. In addition, TKIs show differential ATP-dependent
inhibition profiles on this array. Taken together, we expect that arraybased tumor kinase activity profiling may lead to specific TKIphosphorylation fingerprints for personalized treatment.
Sun, Apr 1, 1:00 - 5:00 PM
863/20 - Tyrosine kinase activity profiling of metastatic malignant melanoma:
Identification of possible therapeutic targets and markers predicting response to therapy
Andliena Tahiri1, Kathrine Roe2, Christian Busch3, Per Eystein Lonning3, Anne H.
Ree1, Vessela N. Kristensen1, Juergen Geisler1. 1Institute of Clinical Medicine, Division
of Clinical Medicine and Laboratory Sciences, University of Oslo, and Akershus
University Hospital, Lørenskog, Norway; 2Oslo University Hospital Radiumshospitalet,
Oslo, Norway; 3Haukeland University Hospital and University of Bergen, Bergen,
Norway Poster Session
PO.ET06.01. Kinase and Phosphatase Inhibitors 1
Abstract
Number:
863
Presentation
Title:
Tyrosine kinase activity profiling of metastatic malignant melanoma:
Identification of possible therapeutic targets and markers predicting
response to therapy
Presentation
Time:
Sunday, Apr 01, 2012, 1:00 PM - 5:00 PM
Location:
McCormick Place West (Hall F), Poster Section 32
Poster
Section:
32
Poster Board
Number:
20
Author
Block:
Andliena Tahiri1, Kathrine Roe2, Christian Busch3, Per Eystein Lonning3,
Anne H. Ree1, Vessela N. Kristensen1, Juergen Geisler1. 1Institute of
Clinical Medicine, Division of Clinical Medicine and Laboratory
Sciences, University of Oslo, and Akershus University Hospital,
Lørenskog, Norway; 2Oslo University Hospital Radiumshospitalet, Oslo,
Norway; 3Haukeland University Hospital and University of Bergen,
Bergen, Norway
Abstract
Body:
Metastatic melanoma is an aggressive form of skin cancer that is highly
resistant to conventional chemotherapy. Thus, novel approaches to
improve the current treatment of advanced melanoma are urgently
needed. Kinases are key regulators of cellular processes, and since nearly
half of the tyrosine kinase complement is implicated in cancer, the
information of kinase activity in tumors give important tumor-specific
information on affected pathways and potential targets for treatment. With
this in mind, we performed tyrosine kinase activity profiling of 26 tissue
samples obtained from patients suffering from metastatic stage IV
melanoma, prior to dacarbazine (DTIC) treatment, using Tyrosine Kinase
PamChip® microarrays. Each sample lysate generated an individual
phospho-substrate signature, representing the state of the kinase signalling
cascades, which allowed us to compare affected targets in responders and
non-responders to DTIC treatment. Further, we attempted to evaluate the
kinase activity in response to BRAF inhibitor, PLX4032 (Vemurafenib),
which has shown dramatic and positive effects in advanced melanoma
tumors, and has recently been approved for the treatment of late stage
melanoma. In this case, incubation was carried out on all samples with
and without PLX4032. Our preliminary results showed that although the
compound is mainly directed against a serine/threonine kinase, BRAF,
PLX4032 also showed inhibitory effects on the activity of multiple
tyrosine kinases. Since the drug is only beneficial for melanoma patients
with BRAF V600E mutations, we compared the activated kinase
pathways in patients with and without this mutation in order to identify
alternative survival pathways in patients with wild type BRAF. The
technology used in this study is based on a unique 3D flow-through
microarray format that allows monitoring of the reaction in real time,
ideal for the characterization of pathways. Each array contained 144
peptide substrates with sites for phosphorylation, representing about 100
different proteins. The use of kinase inhibitors will cause inhibition of
corresponding kinases of activated signalling pathways; hence, providing
us leads to therapeutic targets and possible prediction markers for
response to therapy. The detailed effects of both PLX4032 and sunitinib
in the chosen test system are currently prepared for presentation.
Sun, Apr 1, 1:00 - 5:00 PM
862/19 - Pre-clinical and molecular analysis of Crizotinib in c-Met positive uveal
melanoma Mark J. de Lange, Mieke Versluis, Gre P.M. Luyten, Martine J. Jager, Pieter
A. van der Velden. LUMC, Leiden, Netherlands Poster Session
PO.ET06.01. Kinase and Phosphatase Inhibitors 1
Abstract
Number:
862
Presentation
Title:
Pre-clinical and molecular analysis of Crizotinib in c-Met positive uveal
melanoma
Presentation
Time:
Sunday, Apr 01, 2012, 1:00 PM - 5:00 PM
Location:
McCormick Place West (Hall F), Poster Section 32
Poster
Section:
32
Poster Board
Number:
19
Author
Block:
Mark J. de Lange, Mieke Versluis, Gre P.M. Luyten, Martine J. Jager,
Pieter A. van der Velden. LUMC, Leiden, Netherlands
Abstract
Body:
Uveal melanoma (UM) is an intraocular neoplasm with an annual
incidence of 7 per million. UM originates from melanocytes just like
cutaneous melanoma (CM) and similar to CM, the RAS/RAF/MEK/ERK
pathway is involved in the development of UM. However, not all UM
seem to depend on the activation of this pathway. Loss of ERK signalling
may be correlated with progression as the ERK negative cells are derived
from UM metastasis. In these tumours, cells apparently use a different
pathway to proliferate and survive. In order to identify treatment targets
for metastasis patients we set out to identify the pathway which stimulates
proliferation in the ERK negative UM cells. Growth factor receptor
screening revealed that activation of c-Met or HGF receptor, is inversely
correlated with ERK activation leaving the metastatic UM, halfway
progression to metastasis, with both c-Met and ERK activated. In this
study, we tested the efficacy of c-Met inhibitor Crizotinib and
investigated primary and downstream targets to delineate the role of cMet signalling in UM progression.
Crizotinib was shown to specifically inhibit c-Met positive UM and this
was most clear when cells were grown anchorage independent, in line
with a role in dissemination for c-Met. With phospho-proteomics we
showed that c-Met is the primary target of Crizotinib in UM while FAK
emerges as the prime downstream target. Moreover, Src was shown to be
the downstream kinase that transmits the c-Met signal to both FAK and
ERK in metastatic UM. In UM metastasis, lacking active ERK, Src is
absent and FAK is directly activated by c-Met. Combined, our data
support the use of Crizotinib in late stage UM and reveals altered c-Met
signalling in uveal melanoma progression.