Summer Undergraduate Research Program - Fred Hutchinson ...

Administration of a Novel Chemotherapy Agent in a
Pediatric Brain Tumor Mouse Model
Gregory A. Norris1,2 , Michelle Cook Sangar2 , James M. Olson2 1 The College of Wooster, Wooster, OH; 2 Fred Hutchinson Cancer Research Center, Seattle, WA
Introduction Results
Conclusions
2012 Lee Hartwell Poster Award
1. PD-0332991 significantly decreases retinoblastoma
protein phosphorylation, evidence of targeted cyclindependent
kinase 4/6 inhibition.
PD-0332991 Causes Decreased p-Rb in
ATRT In Vitro
PD-0332291 Causes Decreased p-Rb in Colon
Carcinoma Cells In Vitro
Atypical Teratoid Rhabdoid Tumor (ATRT) is a rare, highly malignant
pediatric central nervous system tumor. The prognosis is poor, with a
2-year survival rate estimated at 15%. 1
2. PD-0332991 is efficacious in vivo, resulting in tumor
regression.
Control 0.062 0.125 0.25 0.5 1 µM
PD-­‐0332991
PD-­‐0332991
0.031 0.062 0.125 0.25 0.5 1 µM
DMSO
Published data shows that approximately 75% of ATRTs possess a
hallmark mutation in the SMARCB1 gene. Loss of SMARCB1 leads to
cyclin D upregulation, a critical protein in cell cycle control. 2,3
3. PD-0332991 is a promising chemotherapy agent for
the treatment of pediatric atypical teratoid rhabdoid tumor.
p-Rb
p-Rb
Rb
Rb
Future Work
α-Tubulin
α-Tubulin
Test PD-0332991 in ATRT mice with tumors in the
brain. Determine whether the drug is able to cross the
blood brain barrier.
Figure 4. Western blot analysis for determining the level of
phosphorlyated retinoblastoma (p-Rb) and total retinoblastoma
protein (Rb) in vitro. ATRT cells from culture were treated with different
concentrations of PD-0332991 for 24 hours.
Figure 3. Western blot analysis for determining the level of phosphorlyated
retinoblastoma (p-Rb) and total retinoblastoma protein (Rb) in vitro. Colo-205
cells from culture were treated with different concentrations of PD-0332991 for 24
hours.
PD-0332991 Suppresses Proliferation
Use flow cytometry to confirm tumor cells are in G1 arrest following cyclin-dependent kinase 4/6 inhibition.
PD-0332991 Causes Decreased p-Rb in ATRT
In Vivo
Ki-67 Staining"
Vehicle Vehicle Drug Drug Control
109
Test PD-0332991 in combination with traditional
cytotoxic chemotherapy agents.
PD-0332991"
Vehicle"
p-Rb
Acknowledgments
Rb
Figure 1. The G1 /S transition. The G1 /S transition is a critical point in the cell cycle at
which cells become committed to cell division. It is regulated by the cyclin-dependent
kinases 4 and 6 (CDK 4/6)—cyclin D—retinoblastoma protein (Rb) signaling pathway. 4,5
α-Tubulin
I would like to thank the Summer Undergraduate
Research Program for this opportunity. The program is
supported in part by the Cancer Center Support Grant
(CCSG) CURE Supplement: 3 P30 CA015704-38S1.
Progression through the G1/S transition requires phosphorylation of
Rb by CDK 4/6 in complex with cyclin D. Rb phosphorylation leads to
the release of bound E2F, a transcription factor responsible for the
regulation of genes that control DNA replication. 4,5
Figure 6. Inhibition of proliferation in ATRT tumors by PD-0332991. Mice bearing ATRT
flank tumors were treated with PD-0332991 for 2 days. Immunohistochemistry analysis was
used to compare the level of Ki-67, a marker of proliferation, between drug-treated and
vehicle-treated tumors.
Figure 5. Inhibition of retinoblastoma phosphorylation in ATRT tumors by PD-0332991.
Mice bearing ATRT flank tumors were treated with PD-0332991 for 2 days. Western blot analysis
was used to determine the level of p-Rb in the tumor lysates.
This work was also supported by the Infant and Toddler
Brain Tumor Grant (R01CA155360) and the Seattle
Children’s Neuro-oncology Endowment Support.
PD-0332991 Treatment Causes Tumor Regression In ATRT Mouse Model
The G1/S phase checkpoint is
dysregulated in ATRTs due to overexpression
of cyclin D1,which leads to
inappropriate retinoblastoma protein
phosphorylation. 3
3000
Special thanks to Michelle Cook Sangar, Chris Hubert,
and the rest of the Olson lab team.
2500
Vehicle111(7L)
Vehicle121(3RR)
Vehicle131(5NP)
Vehicle141(9LL)
Vehicle151(1R)
Drug111(11NP)
2000
References
Figure 2. Molecular Structure
of PD-0332991
1500
PD-0332991, a reversible CDK 4/6
inhibitor developed by Pfizer, was tested in
an ATRT mouse model. An ATRT tumor
fragment from a pediatric patient was
xenografted into the flanks of mice and
passaged. Tumor-derived cell lines were
also propagated in vitro.
1. Cocce et al. A complex karyotype in an atypical teratoid/rhabdoid tumor: case report
and review of the literature. J Neurooncol 104. 375-380 (2011).
Drug121(6R)
Drug131(4L)
Drug141(2RR)
Drug151(8LR)
1000
Tumor*Volume**(mm 3 )
2. Charles W.M. Roberts & Jaclyn A. Biegel. The role of SMARCB1/INI1 in the
development of rhabdoid tumor. Cancer Biol Ther 8. 412-416 (2009).
500
Objectives
3. Biegel et al. The Role of INI1 and the SWI/SNF Complex in the Development of
Rhabdoid Tumors: Meeting Summary from the Workshop on Childhood Atypical
Teratoid/Rhabdoid Tumors. Cancer Res 62. 323-328 (2002).
0
1. Confirm cyclin-dependent kinase 4/6 inhibition after
PD-0332991 treatment.
4. Silvia Lapenna & Antonio Giordano. Cell cycle kinases as therapeutic targets for
cancer. Nature Rev. Drug Discovery 8. 547-566 (July 2009).
1 5 8 11 15 19 22 26 29 32 36 39 43 46 50 53 57 60
Days
2. Determine the efficacy of PD-0332991 therapy in atypical
teratoid rhabdoid tumor.
5. Fry et al. Specific Inhibition of cyclin-dependent kinase 4/6 by PD0332991 and
associated antitumor activity in human tumor xenografts. Mol. Cancer Ther. 3.
1427-1438 (2004).
Figure 7. Long-term efficacy of PD-0332991 treatment in an ATRT patient-derived xenograft mouse model. Mice with ATRT flank tumors were
treated with either PD-0332991 or vehicle daily by oral gavage.
3. Determine whether PD-0332991 warrants further study in
atypical teratoid rhabdoid tumor.
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