**7. The role of autophagy in preventing terminal senescence**

Our further observations in the PA1 model showed the importance of autophagy in withstanding the proteotoxic stress following ETO treatment and its crucial role in maintaining viability; inhibition of autophagy culminated in chromatin fragmentation and nuclei disintegration [46, 57]. Stress-activated OCT4A mainly colocalized, and correlated in its nuclear concentration in individual cells, with activated AMPthr172 kinase (**Figure 5A, B**). AMP-activated protein kinase (AMPK) serves as a general energy depletion sensor and activator of autophagy. The energy stress-response of AMPK is also tightly linked to the DDR of p53 [58, 59] and can induce a p53-dependent glucose-sensitive metabolic checkpoint [51] precipitating apoptotic cell death from the G1/S and likely autophagic death from G2M checkpoint [60].

We found, in addition, that active autophagy in PA1-ETO cells sequestered p16INK4A aggresomes within the autophagic vacuoles (**Figure 5C** i–ii), while disability of autophagy enabled p16 diffuse distribution in the cell nuclei and caused terminal senescence with nuclear disintegration (**Figure 5** iii-iv) preventing survival of ETO-treated cells [46]. The p53-dependent role of AMPK, its relationship with stress-activated OCT4A, and the role of autophagy in the prevention of terminal senescence for TP53 wild-type CSC cells is schematically presented in **Figure 3**.

However, in *TP53* mutants, the cells find an additional pathway for potential recovery – they undergo mitotic slippage and employ polyploidy for repair and sorting the damaged DNA.

**Figure 4.** Population heterogeneity in expression of OCT4A and p21CIP1 in response of PA1 cells to ETO treatment. (**A**) Population heterogeneity in expression of OCT4A and p21CIP1 is extended for days 2–6 post ETO treatment, before recovery from day 7, when it reduces; (**B**) Population heterogeneity is accompanied by a degree of polyploidy as represented by DNA histograms on day 4, with underreplication of the cells in late S-phase of diploid and polyploid cell

cohorts; this phenomenon is enhanced after impediment of autophagic flux. Republished from Ref. [46].

**Figure 5.** Autophagic response of PA1 cells to ETO-mediated DNA damage: (**A**) Scatterplot of image cytometry of OCT4A and pAMPK in individual cells assessed on day 4 after ETO treatment. There is a clear correlation between enhanced expression of OCT4A and pAMPKThr172 of ETO-treated cells; (**B**) OCT4A and pAMPKThr172 immunofluorescence in PA-1 cells 4 days after ETO-treatment. (**C**) The relationship between autophagy and senescence. PA1-ETO cells were treated without or with Bafilomycin A1, prior to media being removed and replaced with fresh media; cells were harvested 48 h later (day 4). (i) imunofluorescent staining for p16ink4a, LAMP2 or DAPI as shown via the BRG optical filter, (ii) only LAMP2 demonstrating high level of functional autophagy sequestrating p16ink4a-containing aggresomes. (iii–iv) shows an example of autophagic failure, where sequestration of p16ink4a is partly lost, diffusing into the cytoplasm and nucleus, which is destroyed. Bars = 10 μm. Republished from Ref. [46].
