SW044248 is selectively toxic for certain NSCLC cell lines. A. IC₅₀ values from 96 h incubation in SW044248 for 46 NSCLC cell lines and 4 immortalized HBEC lines. Cellular ATP content was measured by CelTiterGlo in 384 well format in duplicate. B. Dose-response to 6 day treatment with SW044248 for 8 NSCLC and a HBEC30KT line. Cell viability was measured by uptake of neutral red. C. SW044248 induces apoptosis in HCC4017 but not HBEC30KT. Cells were treated overnight with the concentrations shown. D. 2 μM of SW044248 induces cleavage of PARP beginning after 2 h in HCC4017 cells. E. Structure of SW202742 which has an additional methyl group at the position indicated by the colored circle. F. SW202742 is much less active than SW044248. The dose response of HCC4017 to the two compounds is shown. Cell viability was measured by neutral red after 6 days.

SW044248 rapidly inhibits RNA, DNA and protein synthesis in HCC4017 but not HBEC30KT cells. A. HCC4017 cells were treated for 6 h with the concentrations of SW044248 shown and then labeled for 45 min with Click-IT© (Molecular Probes) reagents for metabolic labeling of RNA (red), DNA (green), or protein (red). Cells were counter stained with DAPI (blue) to label nuclei. B. HBEC30KT cells were treated with 10uM SW044248 for 6 h and labeled as in part A.

SW044248 and CPT inhibit Top1 differentially. A. SW044248 does not inhibit Top2.
Concatenated DNA was incubated with 1% DMSO, 10 μM SW044248, 100 μM CPT, 100 μM etoposide, 10 μM cisplatin, 10 μM cycloheximide and 4U Top2 and electrclonecophoresed on an agarose gel. DNA decatenated by Top2 enters the gel but stays in the loading well when Top2 is inhibited. B. SW044248 inhibits relaxation of supercoiled (SC) DNA in vitro. Concentrations of reagents were 10 μM SW044248, 10 μM CPT, 100 μM etoposide, 10 μM cisplatin, 10 μM cycloheximide and 1 U Top1. C. Inhibition of Top1 by SW044248 increases with increasing concentration. D. Non-toxic compound SW202742 does not inhibit Top1 in vitro. Concentrations are μM. Data are representative of 3 or more experiments. E. 30 min pretreatment of 10 U Top1 with SW044248 blocks CPT-induced uncoiling of supercoiled (SC) DNA. SC DNA was incubated with the agents shown and electrophoresed on an agarose gel. CPT increases nicked open circle DNA levels in presence of 10 U Top1 while SW044248 prevents unwinding of SC DNA. SW044248 pretreatment blocks CPT activity in vitro. F. Treatment with 1 μM CPT but not 5 μM SW044248 decreases cellular Top1 protein in HCC4017 cells. G. Treatment of HCC4017 cells with 10 μM SW044248 from 1 h before addition of 5 μM CPT blocks the decrease in Top1 protein induced by CPT. On the figure the hours indicate the time of chase after adding CPT. H. HCC4017 cells were treated with DMSO, 2.5 μM Camptothecin, or 10 μM SW044248 for 1 h and then embedded in agarose and extracted with detergent in a TARDIS assay to detect Top1 crosslinked to DNA. DNA was stained with Hoechst (blue) and Top1 with antibody (green). All panels of H are the same magnification. The size bar = 400 μm. SC DNA, supercoiled DNA; OC DNA, open circle, relaxed DNA; CPT, camptothecin; SW, SW044248.

SW044248 rapidly activates the integrated stress response through kinases GCN2 and PKR. CPT is much less effective in activating the integrated stress response. A. eIF2α is phosphorylated and inhibited by the four kinases shown. ATF4 translation is enhanced under these conditions. B. Three NSCLC cell lines resistant and 3 cell lines sensitive to SW044248 were treated ± 5 μM SW044248 for 6h. Cell lysates were immunoblotted for eIF2α phosphorylation on Ser51. C. HCC4017 cells were treated with 2 μM SW044248 for the intervals shown, harvested and cell lysates immunoblotted for the proteins shown. D. HCC4017 cells were treated with DMSO, 2 μM SW044248, or 10 μM CPT for 3 or 6 h and cell lysates were analyzed by immunoblotting for the protein shown. E. Cells were pretreated for 2 h with 5 μM CPT and then 5μM CPT ± 10 μM SW044248 for the times shown. Samples were immunoblotted for Top1 and Ser51 phosphorylated eIF2α. The amount of eIF2α phosphorylation is positively correlated with the amount of Top1 remaining. CPT, camptothecin. F. HCC4017 cells were treated for 2 h with 10 μM SW044248, then with either 10 μM SW044248 alone (−) or with 5 μM CPT and 10 μM sW044248 for the times shown.

siRNA knockdown of GCN2 or Top1 decreases the stress responses to SW044248. A. HCC4017 cells were treated with control or individual GCN2 siRNAs for 72 h and then treated with 2 μM SW044248 (+) or DMSO (−) for 6 h. Cell lysates were probed by immunoblotting with antibodies specific for the proteins shown. B. HCC4017 cells were treated with two different siRNAs targeting Top1 or with a control siRNA for 72 hours and then treated ± 2 μM SW044248 for 6 hours. The cells treated with the non-targeting control siRNA received DMSO at the same final concentration as in the compound treated cells. Immunoblots for the proteins are representative of 3 experiments. C. The ATP concentration was measured in cell samples treated with control or GCN2 siRNAs for 72 h, then treated an additional 48 h with 2 μM SW044248 and additional siRNA. D. ATP levels of cells treated as in part A, with Top1 or with a control siRNA for 72 hours and then treated ± 2 μM SW044248 for 48 hours, were measured by Celltiter-Glo. Error bars are standard deviations of the means of 3 samples (data is representative of 4 independent experiments).

Cells resistant to SW044248 increase p21 ^CDKN1A and cells sensitive to SW044248 do not. A. HBEC30KT and HCC4017 cells were treated overnight with the concentrations of SW044248 shown, and lysates were immunoblotted for p21 ^CDKN1A and actin. B&C. HBEC30KT or HCC44 cells were treated with control or siRNAs to p21 ^CDKN1A for 48 h and then treated with DMSO or SW044248 for 48h. ATP was measured with and normalized to the control siRNA treated with DMSO. D. Immunoblot for p21^CDKN1A expression. E. HCC4017 and HCC4017 p21^CDKN1A Clone 5 stably expressing p21^CDKN1A were treated 24 h with the concentrations of SW044248 shown and the phosphorylation of eIF2α and H2AX measured by immunoblotting. F. HCC4017 and HCC4017 p21^CDKN1A clone 5 were treated for 4 days with the concentrations of SW044248 shown and ATP was measured as an indication of cell viability. Graphed data are the means of triplicate samples, with SEM. Data is representative of two or more experiments.