1. Lentiviral Kinase shRNA Screening for Therapeutic Targets in Osteosarcoma - Identifying kinases essential for osteosarcoma cell survival Zhenfeng Duan, Diana Ji, Edward Weinstein, Edwin Choy, David Harmon, Petur Nielson, Andrew Rosenberg, Cao Yang, Joe Schwab, John Iafrate, Ramnik Xavier, Henry Mankin, Francis Hornicek

2. MISSION ® kinase shRNA Library ~ 5 shRNA constructs per gene target  4 within coding sequence of the gene  1 within the 3’UTR for cDNA rescue  Provides high gene coverage  Multiple knockdown levels  Verification of phenotype  Different shRNA produces the same results 5’UTR3’UTRCDS

3. Protein Kinase  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

4. MISSION® LentiExpress™ Human Kinase shRNA  Targets 673 human kinase genes   3,109 lentiviral shRNA clones   Pre-arrayed in 41 ready-to-use 96-well plates   Up to 80 shRNA constructs per plate  5000 Titering Units (TU) per well  Negative controls located on outer columns (1 & 12)

5. TRC shRNA Transfer Vector  Expression vector -pLKO.1 -Lentiviral-based Vector -Puromycin Selection  Promoter -U6 (human)  Design -Broad Institute algorithm -21 bp stem -6 bp loop  5 clones per target gene -Gene coverage -Duplication of phenotype -3’ UTR clone for cDNA rescue

6. Protocol for shRNA targeted kinases screening in human osteosarcoma cell line Dispense KHOS cells into 96-well lentiviral shRNA kinase plates Incubate plate at 37°C, 5% CO 2 Replace cells with fresh media overnight Incubate plate at 37°C, 5% CO 2 Add puromycin at 1µg/ml 7 day duration- Change media every 2 days with puromycin Remove plates Analyze cell proliferation with CellTiter96®AQ ueous One Solution Incubate plate at 37°C, 5% CO 2

7. pLKO.1 Control Particles (C) Non-Target shRNA Control Particles (N) Control Media (M) Negative controls used in the optimization plate 0.5 µg/ml of puromycin causes complete cell death of KHOS, U-2OS and UCH1 in 5 days

8. Representational plate data C: empty vector control N: non-target shRNA control M: media only control

9. Lentiviral shRNA directed against ROCK1 in KHOS cells Absorbance (490nM) pLKO.1 particles Non target particles Media control

10. ROCK1 and cancer  Rho/Rho kinase pathway may be important for cancer invasion, growth, and metastasis. ROCK1 is found to be overexpressed in breast cancer.  Expression of a dominant-negative, kinase-defective ROCK1 mutant significantly inhibited the invasiveness of the cell.  A specific ROCK1 inhibitor, Y-27632, or Wf-536 inhibited invasiveness of several animal and human cancer cells.

11. Expression of ROCK1 in sarcoma U-2OS KHOS MES-SA CS-1 SS-1 TC-71 SKOV-3 3A 2008 ROCK1 Actin 160 kDa A: Cell lines B: Osteosarcoma tissues Actin ROCK1 OST1 OST2 OST3OST5 OST6 OST4

12. Lentiviral shRNA directed against PLK1 in KHOS cells Absorbance (490nM) pLKO.1 particles Non target particles Media control

13. PLK1 and cancer  PLK1 is overexpressed in a broad range of human tumors  Overexpression of either wild-type PLK1 or kinase- inactive PLK1 (mutant) results in multinucleation  Constitute expression of PLK1 causes the transformation of NIH3T3 fibroblasts  P53 protein is phosphorylated by PLK1, which inhibits the pro-apoptotic function of p53

14. Expression of PLK1 in sarcoma MES-SA SKOV-3 U-2OS KHOS CS-1 SS-1 TC-71 3A 2008 PLK1 Actin 63 kDa A: Cell lines B: Osteosarcoma tissues Actin PLK1 OST1OST2OST3OST5OST6OST4

15. PLK1 knockdown induce apoptosis in osteosarcoma cells Apoptosis (O.D)

16. Absorbance (490nM) Scytonemin (µM) PLK1 inhibitor, Scytonemin, inhibits Osteosarcoma cell proliferation and induces apoptosis PLK1 Actin Scytonemin (µM) 0 0.01 0.1 1 5 10

17. Conclusions  A high-throughput screen using a kinase lentiviral shRNA library has identified ROCK1 and PLK1 as potential therapeutic targets in osteosarcoma cells.  Several osteosarcoma cell lines have exhibited decreased cell proliferation upon ROCK1 or PLK1 expression knockdown.  ROCK1 and PLK1 are highly expressed in sarcoma cell lines as well as osteosarcoma tissues, but not in osteoblatst cells.  Knockdown of PLK1 by shRNA or PLK1 inhibitor induces apoptosis in osteosarcoma cells.