Genetic screening for synthetic lethal partners of polynucleotide kinase/phosphatase: potential for targeting SHP-1 depleted cancers

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1 Genetic screening for synthetic lethal partners of polynucleotide kinase/phosphatase: potential for targeting SHP-1 depleted cancers T.R. Mereniuk et al. Supplemental tlmt Material: il Supplemental l Figures 1-7 Supplemental Tables 1 and 2 1

2 A B A549δPNKP A549 PNKP SHP-1 SHP-1 SHP-1 SHP-1 #5 #6 #1 #11 A549 SHP-1 β-actin β-actin C D Karpas pci only Karpas SHP-1 A549 SHP-1 SHP-1 β-actin β-actin Supplemental Figure 1. Western blots of key proteins used during experimentation. (A) Stable knockdown of PNKP in A549δPNKP compared to A549-Scramble control lysate. (B) Transient knockdown of SHP-1 in A549 using four distinct sirnas targeting SHP-1 mrna. (C) Reexpression of SHP-1 in Karpas 299 cells alongside the vector only control (Karpas+pCI) p and a positive control (A549). (D) Level of SHP-1 protein remaining in the stable A549δSHP-1 cell line. 2

3 4 A549-Scramble 35 Replicate # R² = Replicate #1 45 A549δPNKP 4 Replicatte # R² = Replicate #1 Supplemental Figure 2. Comparative analysis of the entire druggable genome sirna screen. Cell viability values from all the plates in the screen were normalized and plotted against their respective replicates. In both the screen using (A) A549-Scramble cells and (B) A549δPNKP cells, the duplicates were shown to have good reproducibility with R2 values of.75 and.759, respectively. 3

4 A549δPNKP 1 A549-Scramble 8 Per rcent survival SHP-1 KCND2 TAP1 sirna used Supplemental Figure 3. Survival of SHP-1 and PNKP co-disrupted cells compared to randomly selected non-hits. There was a large difference in survival between those that were deemed hits when compared to those that were labeled non-hits. Error bars represent standard error (± S.E.) from at least three independent determinations for the SHP-1 values. Error bars for the KCND2 and TAP1 values represent standard error (± S.E.) taken from the raw data from the duplicate screens. 4

5 Supplemental Figure 4. SHP-1 and PARP-1 do not have a synthetic lethal partnership. (A) Clonogenic survival assay showing lack of sensitivity of A549δSHP-1 cells towards the PARP-1 S inhibitor DPQ. Q( (B) Cell proliferation assay following transient transfection of PARP-1 or ASN sirna in A549δSHP-1 or A549-Scramble cells. (C) Western blots showing PARP-1 protein levels following transient transfection with sirna. 5

6 Type 1 Type 2 Type 3 Type 4 Type 5 Supplemental Figure 5. Characteristics of typical comets scored. The tail of the comets indicates the level of DNA damage present in these cells beginning with type 1 comets that showed no DNA damage, progressing to type 5 comets, which showed the most DNA damage (Kumaravel TS, Vilhar B, Faux SP, et al. Comet Assay measurements: a perspective. Cell Biol Toxicol. 29;25:53-64). 6

7 1 Type 1 8 Type 2 Type 3 6 Type 4 4 Type 5 A Perce ent comet ty ype Control Control Control B C Time (h) 7

8 Percent comet typ pe Type 1 Type 2 Type 3 Type 4 Type 5 Control Control D E F Control Time (min) 8

9 Supplemental Figure 6. Single-cell gel electrophoresis (comet) assays for DSBR (A-C) and SSBR (D-F). A549-Scramble, A549δPNKP and A549 cells stably depleted of SHP-1 (A549δSHP-1 cells) were grown to confluence in 6-mm plates in complete DMEM/F12. The cells were irradiated with 5 Gy ( 6 Co Gammacell; Atomic Energy of Canada Limited, Ottawa, Canada) and incubated at 37 C for, 2, 6, and 24 h for neutral comet assays and, 1, 3, 6 or 12 min for the alkaline comet assay. Controls were also included in which cells were not irradiated to give the baseline level of DNA damage present in each cell line. Double and single-strand breaks were then determined by single-cell gel electrophoresis as previously described (Kumaravel TS, Vilhar B, Faux SP, et al. Comet Assay measurements: a perspective. Cell Biol Toxicol. 29;25:53-64; Freschauf GK, Mani RS, Mereniuk TR, et al. Mechanism of action of an imidopiperidine inhibitor of human polynucleotide kinase/phosphatase. J. Biol. Chem. 21;285:2351-6). Each experiment was performed in duplicate, with ten random microscope fields examined per determination. The data were combined to generate a higher total number of cells counted. 2-6 comets were analyzed for each time point. 9

10 h A549-Scramble + 1 μm BH3I-1 4 h 24 h 48 h Supplemental Figure 7. A549-Scramble cells were treated with 1 μm of the apoptotic inducer BH3I-1 continuously over a period of 48 and the phosphorylation of H2AX was monitored at the indicated time points. Quantification of the H2AX phosphorylation (Fig. 7) suggested that only a minor proportion of the H2AX phosphorylation observed when SHP-1-depleted cells are treated with the PNKP inhibitor is due to apoptotic DNA fragmentation. 1

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21 Supplemental Table 2. Tumor suppressors identified as potentially synthetic lethal with PNKP by the sirna screen a These values were obtained with a relatively high concentration (56 nm) of sirna for each target gene, which may explain the poor survival in A549-Scramble cells of some of the possible hits. All subsequent confirmatory experiments were conducted using 2 nm of sirna to reduce toxicity in A549-Scramble cells. 21