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Category:PhysicalandLifeSciences
DegreeLevel:B.S.HealthScience
AbstractID#1048
Validating CDK4 and XPO1 dependencies in a pediatric undifferentiated sarcoma
Bryan Kynnap1,3, Andrew L. Hong2,3,5, Glenn Cowley5, Moony Tseng5, Jamie Cheah5, Mihir Doshi3,5,Coyin Oh5, Alanna Church2, Carlos Rodriguez-Galindo2,3, Katherine Janeway2,3, Kimberly Stegmaier2,3,5, Paul van Hummelen3, Ali Samji5, David Root5,
Jesse Boehm5, William C. Hahn3,4,5
1 Northeastern University; 2 Boston Children’s Hospital, 3 Dana-Farber Cancer Institute, 4 Brigham and Woman’s Hospital, Boston, MA, 5 Broad Institute, Cambridge, MA
Introduction
Results
The intersection between nanomedicine and precision medicine depends
on the understanding of cell pathway mechanisms. Undifferentiated
sarcomas account for up to 20% of all soft tissue sarcomas and therapies
for this type of cancer are ill defined with poor outcomes for those with
metastatic disease. For patients with undifferentiated sarcomas,
understanding of the biology is limited due to its rarity and lack of in-vitro
models. We have developed a novel undifferentiated sarcoma cell line
from a pediatric patient with metastatic disease. This cell line is
genomically silent but carries a number of novel fusions. Through an
orthogonal set of screens utilizing small molecules, shRNAs and
CRISPRs, we identified CDK4 and XPO1 as potential actionable targets.
C.
G.
Figure G. After cells are
treated with KPT-330 for 24
hours, we see Caspase-3
activation and p53
accumulation inside the
nuclear envelope.
Figure B. Validation of knockdown with shRNAs to CDK4 in Peds015T.
Approach
Proof of Concept: Peds015T Undifferentiated Sarcoma
Patient’s clinical course
B.
Results
Cell line’s course
1) Generated from a
metastatic brain lesion in
RPMI media
Figure C. Increased G0/G1 cell cycle arrest in Peds015T and TM87 when compared to
A549 cells when treated with CDK4 inhibition after 24 hours.
D.
H.
2) Rapid growth without
supplements
Figure H. After cells
are treated with
KPT-330 for 24
hours, we see
CRM1/XPO1
inhibitors activating
p53 pathway.
3) Currently at passage
150+
Figure D. Inhibition of CDK4 with LEE011 causes decreased expression of pRb(S807/811).
E.
Results
A.
Figure F. Validation of
shXPO1 knockdown in an
early passage PEDS015T
compared to historical
samples.
nM
A549
796
A204
138
SMS-CTR
A673
Peds015T
274
262
45
TM-87
18
TC-32
23
Figure E. Validation of targeting XPO1
drug sensitivities.
F.
Figure A. Validation of targeting CDK4 drug sensitivities of the highlighted compound in
an early passage of the undifferentiated sarcoma cell line compared to other cell lines (e.g.
rhabdomyosarcoma, Ewing sarcomas and rhabdoid tumors) with IC50 dose curves.
IC50
Conclusions
We have validated in-vitro dependencies to CDK4 and XPO1 using
shRNAs and agents currently in clinical trials such as Palbociclib and
KPT-330. When compared with other poorly differentiated sarcomas (e.g.
rhabdomyosarcoma, Ewing sarcoma) and other poorly differentiated
cancers such as rhabdoid tumors, we see similar correlations.
Interestingly, for KPT-330, we have identified nuclear accumulation of p53
and subsequent activation of its pathway as a potential mechanism for
cell death. In the case where small molecule therapeutics are lacking,
nanotechnology could be employed as a potential delivery of shRNAs.
References
1.  Vanarsdale, T., Boshoff, C., Arndt, K., & Abraham, R. (2015). Molecular Pathways: Targeting
the Cyclin D-CDK4/6 Axis for Cancer Treatment. Clinical Cancer Research.
2.  Fletcher, C. (2013). The evolving classification of soft tissue tumors - an update based on the
new 2013 WHO classification. Histopathology, 2-11.
3.  Ho, Y. (n.d.). The expression of CRM1 is associated with prognosis in human osteosarcoma.
Oncol Rep Oncology Reports.
Acknowledgments
This CaNCURE co-op research project was supported by National Cancer Institute
grant #1CA174650-02 (BK), CureSearch for Children’s Cancer (ALH), and NIH
5K12 HD052896-09 (ALH).