zhenfeng duan massachusetts general hospital center for sarcoma and connective tissue oncology
DESCRIPTION
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. Protein Kinase. - PowerPoint PPT PresentationTRANSCRIPT
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
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
Lentiviral Kinase shRNA library
Protocol for shRNA kinase screen in human osteosarcoma cells
Dispense KHOS cells into 96 well lentiviral shRNA kinase plates
Incubate plate at 37°C,
5% CO2
Replace wells with fresh media
overnight overnight
Incubate plate at 37°C,5% CO2
Add puromycin- supplemented media at 1µg/mL
7 daysChange media every 2 days with puromycin
Remove plates from incubator
Analyze results with a cell proliferation assay kit
Incubate plate at 37°C,5% CO2
Representative plate data
C: empty vector controlN: non-target shRNA controlM: media only control
C*
C C
C
N
N
N
N
M
M
M
M
M
M
M
M
A7 A8 A9 A10 A11
M
DYRK1B
ROCK1
The list of positive hits from the kinase lentiviral shRNA screen in KHOS
Other potential hits from the kinase lentiviral shRNA screen in KHOS
KHOS cells control
DYRK1B lentiviral shRNA particles (NM_004714.x-442s1c1)
DYRK1B rescue lentiviral shRNA particles(TRCN0000002140)
KHOS/DYRK1B cDNA+ DYRK1B lentiviral shRNA particles
KHOS/DYRK1B cDNA+DYRK1B rescue lentiviral shRNA particles
Ab
sorb
ance
Ab
sorb
ance
DYRK1B lentiviral shRNA particles (NM_004714.x-442s1c1)
DYRK1B lentiviral shRNA particles (NM_004714.x-2251s1c1)
DYRK1B lentiviral shRNA particles (NM_004714.x-778s1c1)
DYRK1B lentiviral shRNA particles (NM_004714.x-559s1c1)
DYRK1B lentiviral shRNA particles (NM_004714.x-1353s1c1)
pLKO.1 vector lentiviral shRNA particles
Non target lentiviral shRNA particles
Media control
Results of lentiviral shRNA directed against DYRK1B
DYRK1B Control
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
U-2OS
U-2OS DYRK1B shRNA
Saos
Saos DYRK1B shRNA
OSA344
OSA344 DYRK1B shRNA
Osteoblast (HOB-c)
Osteoblast (HOB-c) DYRK1B shRNA
U-2OS Saos OSA344
Osteoblast (HOB-c)
Control
DYRK1BshRNA
Ab
sorb
ance
Knockdown DYRK1B decreases cell proliferation in multiple cell lines
Pro
life
rati
on
(O
.D)
KHOS
KHOS/non-specific siRNA
KHOS/ DYRK1B siRNA
Post-transfection (days)
Post-transfection (days)
Ap
op
tosi
s (O
.D)
Synthetic siRNA targeting DYRK1B decreases cell proliferation and induces apoptosis
DYRK1B 10×
Medium-staining High-staining Low-staining
DYRK1B 40×
Survival proportions
0 50 100 150 2000
20
40
60
80
100 Low-stainingMedium-stainingHigh-staining
Months
Per
cent
sur
viva
l
survivor non-survivor
0
1
2
3
Mir
k st
aini
ng 2.2
1.5
P=0.0012P=0.001
DYRK1B expression is correlated with poor osteosarcoma survival
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
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
U-2
OS
KH
OS
MES
-SA
CS-
1
SS-1
TC-7
1SK
OV-
33A 20
08
DYRK1B
Actin
75 kDa
OST
1O
ST2
OST
3O
ST4
OST
5O
ST6
HO
B-c
NH
Ost
hFO
B
DYRK1B expression in tumor cell lines and osteosarcoma tissues
DYRK1B
Actin
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
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