1. IDENTIFICATION OF DUAL-SPECIFICITY TYROSINE-(Y)-PHOSPHORYLATION REGULATED KINASE 1B (DYRK1B) AS A POTENTIAL THERAPEUTIC TARGET IN OSTEOSARCOMA
Zhenfeng Duan
Massachusetts General Hospital
Center for Sarcoma and Connective Tissue Oncology

2. Protein Kinase
A type of enzyme that transfers phosphate groups from ATP, to specific substrates. The process is referred to as phosphorylation
One of the largest family of genes in human genome
Constitutes about 2% of human genes
Phosphoproteins represent about 30% of cellular protein
More than 400 human diseases are associated with kinase signaling
Over 30% of all research spending on drug development focuses on kinases

3. Lentiviral Kinase shRNA library

4. Analyze results with a cell proliferation assay kit
Protocol for shRNA kinase screen in human osteosarcoma cells
Analyze results with a cell proliferation assay kit
Dispense KHOS cells into 96 well lentiviral shRNA kinase plates
Remove plates
from incubator
Replace wells with fresh media
Add puromycin- supplemented media at 1µg/mL
7 days
Change media every 2
days with puromycin
overnight
overnight
Incubate plate at 37°C,
5% CO2
Incubate plate at 37°C,
5% CO2
Incubate plate at 37°C,
5% CO2

5. Representative plate data
DYRK1B C* A7 C A8 A9
A10
A11 C C N N
ROCK1 N N M M M M M M M M M
C: empty vector control
N: non-target shRNA control
M: media only control

6. The list of positive hits from the kinase lentiviral shRNA screen in KHOS
Other potential hits from the kinase lentiviral shRNA screen in KHOS

7. Absorbance Absorbance
Results of lentiviral shRNA directed against DYRK1B
DYRK1B lentiviral shRNA particles (NM_ x-442s1c1)
DYRK1B lentiviral shRNA particles (NM_ x-2251s1c1)
DYRK1B lentiviral shRNA particles (NM_ x-778s1c1)
Absorbance
DYRK1B lentiviral shRNA particles (NM_ x-559s1c1)
DYRK1B lentiviral shRNA particles (NM_ x-1353s1c1)
pLKO.1 vector lentiviral shRNA particles
Non target lentiviral shRNA particles
Media control
DYRK1B
Control
KHOS cells control
Absorbance
DYRK1B lentiviral shRNA particles (NM_ x-442s1c1)
DYRK1B rescue lentiviral shRNA particles(TRCN )
KHOS/DYRK1B cDNA+ DYRK1B lentiviral shRNA particles
KHOS/DYRK1B cDNA+DYRK1B rescue lentiviral shRNA particles

8. Biology of DYRK1B
Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1B, Minibrain-related kinase; MIRK
Location:19q12-q13.1
Function: Dual-specificity kinase which possesses both serine/ threonine and tyrosine kinase activities. Enhances the transcriptional activity of TCF1/HNF1A and FOXO1.
DYRK1b regulates MEF2-dependent transcription by phosphorylating class II histone deacetylases and reinforces G0 arrest of myoblasts by phosphorylating the cell-cycle regulators cyclin D1, cyclin D3 and p27Kip1

9. Knockdown DYRK1B decreases cell proliferation in multiple cell lines
U-2OS
U-2OS DYRK1B shRNA
Saos
Saos DYRK1B shRNA
Absorbance
OSA344
OSA344 DYRK1B shRNA
Osteoblast (HOB-c)
Osteoblast (HOB-c) DYRK1B shRNA
U-2OS
Saos
OSA344
Osteoblast (HOB-c)
Control
DYRK1B
shRNA

10. KHOS/non-specific siRNA
Synthetic siRNA targeting DYRK1B decreases cell proliferation and induces apoptosis
KHOS
KHOS/non-specific siRNA
KHOS/ DYRK1B siRNA
Proliferation (O.D)
Post-transfection (days)
Apoptosis (O.D)
Post-transfection (days)

11. DYRK1B expression is correlated with poor osteosarcoma survival
2.2
1.5
P=0.001
P=0.0012
Low-staining
Medium-staining
High-staining
DYRK1B 10×
DYRK1B 40×

12. Conclusions
A high-throughput screen using a kinase lentiviral shRNA library has identified DYRK1B as potential therapeutic targets in osteosarcoma cells
Several osteosarcoma cell lines have exhibited decreased cell proliferation upon DYRK1B expression knockdown
DYRK1B are highly expressed in sarcoma cell lines as well as osteosarcoma tissues, but not in osteoblast cells
DYRK1B expression in tumors is closely correlated with poor prognosis in osteosarcoma patients
DYRK1B may serve as a promising target for molecular therapy in the treatment of osteosarcoma

13. MGH Sarcoma Molecular Biology Laboratory
Acknowledgements
MGH Sarcoma Molecular Biology Laboratory
Francis Hornicek
Edwin Choy
Henry Mankin
Cao Yang
Keinosuke Ryu
Michiro Susa
Dexter Liu
Joe Schwab
MGH Pathology
Andrew Rosenberg
Petur Nielsen
Support
Gattegno and Wechsler funds
Sarcoma Foundation of America (SFA)
Sigma-Aldrich Corporation

14. DYRK1B expression in tumor cell lines and osteosarcoma tissues
KHOS
MES-SA
TC-71
SKOV-3
U-2OS
CS-1
SS-1 3A
2008
HOB-c
NHOst
hFOB
DYRK1B
75 kDa
Actin
OST1
OST2
OST3
OST4
OST5
OST6
DYRK1B
Actin

15. DYRK1B/Mirk and cancer
DYRK1B/Mirk is amplified within pancreatic cancer and ovarian cancer cell lines known to exhibit the 19q13 amplicon. Depletion of Mirk has been shown to lead to apoptosis in pancreatic cancer cell lines
Depletion of DYRK1B/Mirk in two rhabdomyosarcoma cell lines and in two pancreatic cancer cell lines, decreased the clonogenicity of these tumor cell lines
Mirk/Dyrk1B is a downstream effector of oncogenic K-ras in pancreatic cancer
Mirk/dyrk1B was found to be one of the four most promigratory genes in highly motile SKOV3 tumor cells by an RNAi screen of >5,200 genes

16. DYRK1B/Mirk and cancer
DYRK1b/Mirk is highly expressed and activated, such as in rhabdomyosarcoma cells, some colon carcinoma cells and HeLa cervical carcinoma cells
DYRK1b/Mirk is expressed at low levels in most normal tissues
DYRK1b/Mirk knockdown by synthetic duplex RNAis enhances response to gemcitibine