Supplemental Information

From

Loss of Function Screen in Rhabdomyosarcoma Identifies CRKL-YES as a Critical Signal for Tumor Growth

Choh L. Yeung, Vu N. Ngo, Patrick J. Grohar, Fernanda Arnaldez, Afua Asante, Xiaolin Wan, Stephen M. Hewitt, Javed Khan, Chand Khanna, Louis M. Staudt, and Lee J. Helman

Supplemental experimental procedures

Supplemental figure legends

Supplemental references

Supplemental Experimental Procedures

In vitro confirmation experiments

shCRKL cell lines were created for validation by infecting RD and Rh30 with retrovirus encoding the shRNA targeting CRKL at bp948 of NM_005207 (GGTGAGATCCTAGTGATAATA) or at bp1305 (GTCACAAGGATGAATATAAAT). RD control and Rh30 control cells were generated using retrovirus encoding an shRNA targeting a region of GFP at bp 488.

CRKL overexpression

CRKL cDNA lacking the 3’ untranslated sequence was cloned from an Invitrogen Ultimate ORFCard (Clone ID IOH26401) template with PCR primers at the 5’ ATG start and at the 3’ stop codon. The product was Topo TA cloned into the pcDNA3.1/V5His expression vector (Invitrogen). Clones were sequenced to verified for integrity and orientation. CMV CRKL cDNA (2ug) was transfected into RD and Rh30 cell lines using the Amaxa Nucleofector (Lonza) (buffer V, program B032) using 2 million cells creating the rescue cDNA cell lines. Vector control cell lines were created in the same manner using pcDNA 3.1/lacZ/V5His. Cell lines were batch selected in G418 (Invitrogen) for 4 weeks.

Cell cycle analysis

Cells were plated at one million cells per 10cm plate overnight then serum starved for 6 or 24 hours before staring doxycycline treatment. Cells were harvested 3-5 days post doxycycline induction for propidium Iodide (PI) staining and FACS. Cells were washed with cold PBS (Invitrogen), scraped and re-suspended in cold PBS and fixed with 70% cold ethanol. Cells stored in 4C overnight then washed and stained with 500ul PI solution (0. 1 % (v/v) Triton X-100 (Sigma) in PBS add 2 mg DNAse-free RNAse A (Sigma) and 0.40 ml of 500 µg/ml PI (Sigma)). Cells were incubated at 370C for 15 min and then analyzed.

In vivo xenograft growth

Protocols for animal care were reviewed and approved by the National Cancer Institute Animal Care and Use Committee. A cohort of 10, female, Nude scid-beige mice (Charles River Laboratories) were included in each group of CMV CRKL, shControl, and shCRKL. In the RD and Rh30 xenograft experiments, all the groups were fed with doxycycline diet (Bio-Serv, Frenchtown, NJ) except the initial Rh30 cohort was not fed doxycycline diet to serve as un-induced control. Three million cells were injected into the left gastrocnemius muscle of each mouse. Tumor size was measured in one dimension and mice were sacrificed when tumors reached 2 cm in the largest dimension.

In vivo dasatinib xenograft experiments were done with 20 4 week old, female, mice per cohort per cell line. One cohort of mice was treated with 80mM sodium citrate buffer pH 3.1 and another cohort of mice was treated with dasatinib in 80mM citrate buffer pH 3.1 at 100mg/Kg. Mice were treated once a day times 6 days per week (six days of treatment, one day off) by gavage for up to 52 days.

At the end of the experiment tissue samples were collected from tumor-bearing mice, immediately frozen on dry ice, and stored frozen at -80oC until used for Western blot analysis. 30µg of total protein per tumor tissue sample were loaded per lane. Tumor volume was calculated using the formula: (Maximum diameter x 2(minimum diameter)). All mice were euthanized by CO2 asphyxiation when the tumor reached 2 cm in the largest dimension.

Western blot for Xenograft tissue

Western blot analysis of tumors was performed after homogenizing 0.5g of tumor tissue in 5ml of T-PER (Pierce, Rockford, IL). BCA protein assay (Pierce) was used to determine total protein for gel loading. 30µg of total protein per tumor tissue sample were loaded on a NUPAGE gel and tumors were analyzed for pCRKL in both tetracycline treated and control animals.

SFK inhibition assays

Cell proliferation was measured using CellTiter 96 AQueous one solution (MTS) assay (Promega). RMS cells were plated at 1000-3000 cells per well in a 96 well plate and incubated overnight at 37oC, 5% CO2. The cells were then treated with either 250nM dasatinib, or 5µM saracatinib, or 125-250 nM crizotinib, with DMSO as vehicle control for 72 hours. Each treatment group was a cohort of six wells. The assays were performed according to Promega’s protocol and the plate was read at 1 hour after addition of reagent on a Molecular Device VERSAmax plate reader. One thousand cells per wells was use for the dasatinib and crizotinib proliferation assay and three thousand cells per wells for the saracatinib.

ShYES lentivirus

Cells were plated at 1000 cells per well and 5:1 lentivirus particles to cell ratio was used for transduction of shYES, with 5µg/ul of polybrene. 24 hours post infection the media was replaced with fresh media and 72 hours post infection the media was replaced with media containing 5ug/ml of puromycin. MTS assay was read when shRNA control was near 50-70% confluence or 11 days post infection. Lentivirus shYES clone 9 and 11 was from Sigma (TRCN0000001609 and TRCN0000001611, respectively).

Serum free and growth factor depravation

Cells were plated in 10 cm plate overnight for Western analysis as described. Complete media was removed, cells were washed with PBS and were replated in serum-free or serum containing media or in 10% charcoal striped serum media (Invitrogen) overnight. The cells were harvested for Western analysis as described.

Transduction of shRNA barcode library

Barcoded, shRNA expression constructs targeting approximately 5000 genes, with each gene was represented by three shRNAs were pooled into a single plasmid prep as previously described (Ngo et al 2006), and 5 plasmid prep pools were combined to make high titer ecotropic viral stocks for infection. RD and Rh30 cells, expressing the TETR, were then infected with the shRNA viral stock. Six days after puromycin selection, half of the cells (1-2 million cells) were treated with 25 ng/mL of doxycycline and the other half were placed into control media. Cells were then incubated for 21 days, after which, genomic DNA was prepared from both doxycycline treated and control cells with a QIAamp DNA mini kit (Qiagen, Valencia, CA). 2 µg of genomic DNA was used as the PCR template to amplify barcode sequences using the primer pairs 5’CTAATACGACTCACTATAGGGAGATGTCGCTATGTGTTCTGGGAAATCAC 3’,

and 5’GGTTAAGATCAAGGTCTTTTCACCTGGC3’ . Barcode amplified sequences were in vitro transcribed using MAXIscript T7 kit Ambion (Invitrogen). One µg of the transcribed products was fluorescently labeled with either Cy3 or Cy5 using a kit from Kreatech (Durham, NC). In vitro transcribed, labeled bar-code RNA was hybridized to bar code microarrays as previously described (Berns et al 2004). Hybridization results were read on a GenePix 4000 array reader (Molecular Device, Sunnyvale, CA).

Retroviral Packaging and tranduction

A mixture of shRNA retroviral constructs containing the retroviral helper plasmids, pHIT/EA6x3*, pHIT60 (kindly provided by Stephen Goff (Markowitz et al 1988), and Lipofectamine 2000 reagent (Invitrogen) were packaged in the human embryonic kidney producer cell line, 293T. 72 hr retroviral supernatant was used to infect doxycycline-inducible RMS cell lines with 8 µg/mL polybrene. Lymphoma cell lines were transduced by spin infection as described (Ngo et al 2006). Puromycin (1µg/mL, Sigma) was used to batch select for stable integrants over 6 days.

Cell proliferation assay

Three independent methods for measuring cell proliferation were done and all showed similar results. RMS cells were plated at 1000-3000 cells per well in a 96 well plate and incubated overnight at 37oC, 5% CO2. The cells were then treated with and without doxycycline, 25 ng/ml for time course assays. Each treatment group was done in groups of 6. Cell proliferation was determined by MTS according to the manufacturer’s recommendations (Promega). The plate was read at 1 hour on a Molecular Device VERSAmax plate reader. For cell confluence, the IncuCyte uses an integrated confluence algorithm as a surrogate for cell number. Cells were plated at 1000 cells per well in 96 wells plates. 24 wells were used as untreated control and another 24 wells were used for doxycycline induction. Cell proliferation was monitored and recorded with the Incucyte system (Essen BioScience). For ATP proliferation assay, 3000 cells were plated per well in a 96 well plate and assayed for ATP according to the manufacturer’s recommendations (Promega).

Supplemental Reference

Berns K, Hijmans EM, Mullenders J, Brummelkamp TR, Velds A, Heimerikx M et al (2004). A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428: 431-437.

Capdeville R, Buchdunger E, Zimmermann J, Matter A (2002). Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug. Nat Rev Drug Discov 1: 493-502.

Cui JJ, Tran-Dube M, Shen H, Nambu M, Kung PP, Pairish M et al (2011). Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK). J Med Chem 54: 6342-6363.

Markowitz D, Goff S, Bank A (1988). A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol 62: 1120-1124.

Ngo VN, Davis RE, Lamy L, Yu X, Zhao H, Lenz G et al (2006). A loss-of-function RNA interference screen for molecular targets in cancer. Nature 441: 106-110.

Yamazaki S, Skaptason J, Romero D, Lee JH, Zou HY, Christensen JG et al (2008). Pharmacokinetic-pharmacodynamic modeling of biomarker response and tumor growth inhibition to an orally available cMet kinase inhibitor in human tumor xenograft mouse models. Drug Metab Dispos 36: 1267-1274.

Zou HY, Li Q, Lee JH, Arango ME, McDonnell SR, Yamazaki S et al (2007). An orally available small-molecule inhibitor of c-Met, PF-2341066, exhibits cytoreductive antitumor efficacy through antiproliferative and antiangiogenic mechanisms. Cancer Res 67: 4408-4417.

Figure Legends:

Figure S1. CRKL overexpression in RMS cell lines transfected with CRKL expressing vector.

CRKL transfected into the RMS cell lines show overexpressed of CRKL in both the RD and Rh30 cell lines compared to their empty vector control (EV).

Figure S2. RD and Rh30 cells lines were G1 arrested with CRKL knockdown. shCRKL induction resulted in RMS cell lines rrested in G1.

RD (A) and Rh30 (B) cells treated with doxycycline, shCRKL induced, lead to increased cell number in G1 phase by 14 percent on day 3 and 7 percent on day 5, respectively, compared to their shCRKL uninduced control. Cells were harvested and stain with propidium iodine (PI) for cell cycle analyzed by FACS.

Figure S3. shCRKL knockdown of CRKL in xenograft tumors.

(A) Western analysis for pCRKL expression in RD xengraft tumors samples demonsrates most tumors in the shCRKL induced group show some loss of CRKL expression compared to the shGFP induced group. (B) pCRKL expression in the shCRKL induced Rh30 xenograft tumors also show partial loss of pCRKL in all four samples compared to the four shGFP induced tumors. Xenograft tumors were harvested when the tumor reached 2CM in the largest dimension.

Figure S4. CRKL and SFK phosphorylation were unchanged by IGF1R blockade.

(A, B) Western analysis showed no sustained alteration of phospho-CRKL and phospho-SFK when RMS cells were treated with a human antibody targeting IGF1R,100nM (R1507,Roche), compared to controls. (C) R1507 decreased phospho-AKT (Ser473) in both RD and Rh30 compared to media control indicating the R1507 was active.

Figure S5A-B. Crizotinib, an inhibitor of MET and ALK, did not alter CRKL phosphorylation, SFK phosphorylation, and did not attenuate RMS cell growth.

(A) Western blot demonstrates that crizotinib (Cui et al 2011, Yamazaki et al 2008, Zou et al 2007)at two different doses does not alter phosphorylation of CRKL and SFK in ERMS (RD) and ARMS (Rh30) compared to their controls. 30ug of total protein was loaded per lane and beta actin was used as a loading control. (B) RMS cell growth was not inhibited by two different doses of crizotinib in ERMS (RD) and ARMS (Rh30) compared to their controls. The p value shown was compared to their respective controls.

Figure S6. Rapamycin has no affect on phosphorylation of CRKL.

RD and Rh30 cell lines with shCRKL induction (doxy) decreased CRKL expression and decreased pY416SFK but did not alter phopho-AKT or phospho-S6 compared to rapamycin where decreased pS6 was detected. Rapamycin did not modifiy pCRKL in either RD or Rh30 cells. Rapamycin also had no effect on pSFK in RD cells but did appear to decrease pSFK in Rh30 cells.

Figure S7. SFK expression in the presence and absent of CRKL knockdown.

CRKL knockdown in RD and Rh30 cells does not affect total protein expression of FYN (A), SRC (B), or LYN (C), but modestly decreased YES protein expression (A).

Figure S8. Saracatinib decreases phospho-Y207-CRKL and RMS growth

(A) Phosphorylation of CRKL Y207 decreases in a dose dependent manner with sarcatinib treatment while total CRKL was unchanged in a two hours treatment, for both RD and Rh30. (B) Inhibition of phospho-Y207-CRKL, phospho-Y416-SFK, and inhibits growth of RD cells (C) and Rh30 cells (D) by 72 hr with 5uM of saracatinib.

Figure S9. Imatinib does not inhibit phosphorylation of CRKL in RD cell line. Used the published IC50(Capdeville et al 2002), 250nM, for ABL inhibition with imatinib (IM), tyrosine phosphorylation of CRKL was not affected compared to DMSO control, whereas 100nM and 200 nM of dasatinib (Das) inhibited phosphorylation of CRKL.

Figure S10. CRKL phosphorylation remains unaltered by serum starvation or growth factors depravation in RMS.

ERMS cell lines (RD and Rh18) and ARMS cell lines (Rh28 and Rh30) were cultured in serum starved (serum free) or growth factors deprived (charcoal stripped serum) serum overnight. Western analysis showed no changes in pCRKL compared to their respective control in10% serum (complete media).

Table S1. Result from the shRNA library screen of RMS cell lines after filtering out lymphoma genes.

Table showed 40 genes that RMS cell lines dependent on for survival identified by the barcoded shRNA library. The 40 candidates were singled out after selection for shRNA that inhibited RMS growth greater than two fold and after subtracting out shRNA that were common to four lymphoma cell lines, screened with the same shRNA library. The table showed fold changes in log 2 for RD and Rh30 cell lines and their corresponding p values.

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