Summer Undergraduate Research Program - Fred Hutchinson ...

Spindle-Checkpoint targeted therapy for Glioblastoma
Woong Hwang1,2 , Chris Hubert2 , Patrick Paddison2 , and James Olson2 1 University of Rochester, Rochester, NY, 2 Fred Hutchinson Cancer Research Center, Seattle, WA
BrdU immunostaining
3.Bromodeoxyuridine (BrdU) immunostaining
Analysis of DNA replication.
DNA replication after Aurora B inhibition
Added BrdU at different time points for 4 hrs.
60
50
40
30
20
10
0
BrdU
DAPI staining to detect total number of nuclei.
%BrdU/DAPI
Negative Control: No BrdU addition.
Primary
antibody
+
CB660 +DMSO
CB660 +AZD
333nM
G166 +DMSO
CB660 +AZD
333nM
Secondary
antibody
0hr 24hr 48hr 72hr 96hr
Drug-treated Time
ABSTRACT
Glioblastoma is the most common and lethal sub-type of primary brain tumor.
Median survival from the time of diagnosis is 14 months, with less than 5% of
patients surviving 5 years. Hence, a new approach to the treatment of glioma needs
to be developed. A recent screening collaboration between the Olson and Paddison
labs has identified the spindle assembly checkpoint as a potential vulnerability in
Glioblastoma. Using primary human tumor cultures, our laboratory has tested
clinically relevant spindle assembly checkpoint inhibitors to determine an effect on
Glioma Stem Cells(GSC) and normal Neuronal Stem Cells (NSC). Aurora Kinase
B inhibitors induced more cellular multinucleation and polyploidy in GSC than in
NSC. Furthermore, Aurora B inhibition uncovered the ability of GSCs to continue
DNA synthesis after cell cycle arrest. Although Aurora B kinase inhibition alone
does not induce apoptosis in GSCs, its ability to unmask specific DNA replication
differences in GSC may make it an important tool to explore glioma biology and
raise a potential role in combination therapies.
2010 Best Poster Award
RESULTS & DISCUSSION
NSC DM SO GSC DM SO NSC AZD 0.33uM GSC AZD 0.33uM
CB660(NSC)
G166(GSC)
Drug Serial Dilution/alamarBlue Assay
48hr GNS AZD
48hr NSC AZD
1.2
1
1.2
1
1.2
1
0.8
0.8
0.8
0.6
0.6
0.6
BACKGROUND
BrdU-GFP
0.4
0.4
0.2
0.2
0.4
0.2
0
Normalized alamarBlue
alamarBlue
0
0
0.004 0.012 0.037 0.111 0.333 1.000 3.000 9.000
0.000 0.001 0.004 0.012 0.037 0.111 0.333 1.000
0.001 0.004 0.012 0.037 0.111 0.333 1.000 3.000
AZD dose ( uM)
ZM dose (uM)
ZM 437439 (uM) AZD-1152 HQPA (uM (uM) VX-680 (uM)
Metaphas
e
Chromosome
Chromosome
Misalignment
Misalignment
No significant difference between drug treated GSC and NSC in metabolic activity.
AlamarBlue data did not reflect viable cell numbers under microscope.
Spindle
assembl
y check
point
Auror
a B
156
Anaphase
No
Biorientation
DAPI
Cells treated with ZM437439, AZD-1152HQPA and VX680 shared similar morphologies
suggesting drugs’ specificity to Aurora B.
Drug
In mitosis, the Spindle Assembly Checkpoint detects an unattached kinetochore. It delays
anaphase onset until all chromosomes correctly align on the microtubule spindle apparatus.
Aurora B inhibited GSCs were able to undergo DNA synthesis even after 96 hrs suggesting
abnormal DNA replication programs
Aurora B kinase is overexpressed in malignant glioblastoma suggesting Aurora inhibitors as
potential therapeutic agents.
Aurora B inhibited NSC did not significantly continue DNA synthesis after 24hrs
suggesting mitotic arrest.
Aurora B kinase plays an essential role in chromosome alignment, bipolar orientation and
cytokinesis.
CONCLUSION
AZD1152-HQPA, VX680, and ZM437439 are selective Aurora B Kinase Inhibitors.
1. Three different drug treated cells shared similar morphological patterns
suggesting the specificity of drugs as Aurora B kinase inhibitors.
DAPI in blue
METHODS
2. Aurora B inhibition allowed prolonged DNA replication without cytokinesis
resulting in multinucleation in glioma stem cells (GSC).
Neural Stem Adherent cell culture
Primary human glioma stem cells and karyotypically
normal neural stem cells were used.
3. Aurora B inhibition allowed limited DNA replication without cytokinesis in
neuronal Stem cells (NSC) .
Drug treatment led to an increase in nuclear volume and cell size in four GSC lines and NSCs.
All 4 glioma lines tested showed bulky abnormal nuclei whereas NSCs showed organized dual
Allow for uniform exposure to growth factors, oxygen
and drugs.
4. The increase in polyploidy corresponded with an increase in nuclear volume and
cell size in both GSCs and NSCs.
nuclei.
5. Aurora B inhibition alone did not induce apoptosis in GSCs
Active Caspase 3/7 Assay
FUTURE DIRECTION
Limit differentiation effects.
Reduced apoptosis.
GSC cultured cells better represent patient tumors than
traditional cell culture GBM lines
Pollard, Cell Stem Cell 2009
Apoptosis resulting Caspase from 3/7 assay Aurora B inhibition
2.5
1. Measure DNA content of drug treated cells by FACS sorting to quantify polyploidy.
2. Confirm BrdU findings in additional normal and glioma lines.
alamarBlue assay Caspase 3/7 assay
2
NSC 48hr
GSC 48hr
1.5
3. Combination drug treatment that targets DNA replication.
4. Investigate DNA licensing defects in GSC lines
1
0.5
Caspase 3/7 activity (R.L.U)
ACKNOLWEDGEMENT
0
AZD 37uM AZD111uM AZD333uM + ctrl DMSO
Drug conditions
This program is supported by the Continuing Umbrella of Research
Experience CURE) [3 P30 CA015704-36S2] and [5 P30 CA015704-35S1].
Thanks for the mentorship from Chris Hubert.
Positive control: Cisplatin+Etoposide+cyclosporin
Caspase activity measured at different time points
Aurora B inhibition alone did not induce apoptosis in GSCs
(24hr, 48hr and 72hr)