Zhenfeng Duan Massachusetts General Hospital Center for Sarcoma and Connective Tissue Oncology

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






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

Documents

Zhenfeng Duan Massachusetts General Hospital Center for Sarcoma and Connective Tissue Oncology