Fig. 2. Chronic DT-061-Induced ATF4-CHOP activation triggers cancer cell death independently of PERK activity and eIF2α phosphorylation.

A Western blotting analysis evaluating the effect of DT-061 treatment on eIF2α phosphorylation, ATF4, and CHOP expression in multiple HGSC and FT lines to evaluate dependency profiles. OV81, PEO-1, PEO-C4.2, and CAOV3 represent HGSC models (shades of orange) while FT246 and FT237 represent non-malignant fallopian tube tissues (shades of green). B Schematic representing the two well-established canonical pathways that phosphorylate eIF2α resulting in ATF4 and CHOP increased expression: PERK (via the UPR) and PKR, GCN2, and HR (as part of the Integrated Stress Response (ISR)). Chemical inhibitors utilized to test PERK and eIF2α contribution to DT-061-mediated ATF4 and CHOP activation are also represented. C Western blot analysis assessing the role of PERK activation and subsequent UPR regulation in OV81 and FT246 cells in the presence of DT-061. Cells were pre-incubated with either DMSO or 1 µM of PERK inhibitor (PERKi) for 3 h. Subsequently, DT-061 (DMSO pre-incubated), fresh PERKi, or the combination of the two drugs (PERKi pre-incubated) were added and cells were harvested after 3, 6, and 24 h of the second round of drug exposure. PERK pathway activity was measured by assessing its hyperphosphorylation state (band shift) and downstream targets’ expression. D Western blot analysis assessing the role of p-eIF2α downstream signaling and its impact in the activation of ATF4 and CHOP proteins in OV81 cells after treatment with DT-061. Cells were pre-incubated with either DMSO or 1 µM of ISR Inhibitor (ISRIB) for 3 h. Cells were then treated with either Tg or DT-061 after both DMSO and ISRIB pre-incubation and harvested at 6 h post second treatment round (total of 9 h). Expression of ATF4 and its downstream targets were evaluated under each of these treatment conditions and quantified (in Supplementary Fig. 2R). E A pool of siRNAs was used to knockdown PP2A-Aα protein expression for 24 h. Western blotting analysis was used to assess cytoplasmic versus nuclear localization as well as expression of the ATF4 and its downstream target genes after DT-061 treatment in both siControl (negative control) and siPP2A-Aα. F Whole cell lysate of the conditions previously described in (E) was also collected to evaluate the expression of cell death markers and ER-associated autophagy. Western blotting analysis was performed (top) and represented as z-score values in a heatmap obtained through western blot quantification (bottom). Calculated z-scores were obtained from protein quantification of three independent biological replicates. G PP2A chemical inhibitor calyculin A, was used to evaluate DT-061’s anticancer effects and their dependency on PP2A in other HGSC, lung, prostate, breast, and colorectal lines. Cells were pre-incubated with either DMSO or 5 nM of calyculin A for 1 h.