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

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

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

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  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. 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 Add puromycin- supplemented media at 1µg/mL Replace wells with fresh media 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* C A7 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. Results of lentiviral shRNA directed against DYRK1B 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) Absorbance DYRK1B lentiviral shRNA particles (NM_004714.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_004714.x-442s1c1) DYRK1B rescue lentiviral shRNA particles(TRCN0000002140) KHOS/DYRK1BcDNA+ 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. 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 • Francis Hornicek • Edwin Choy • Henry Mankin • Cao Yang • Keinosuke Ryu • Michiro Susa • Dexter Liu • Joe Schwab • MGH Pathology • Andrew Rosenberg • Petur Nielsen Acknowledgements • Support • Gattegno and Wechsler funds • Sarcoma Foundation of America (SFA) • Sigma-Aldrich Corporation

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