Background: Cells respond to stress in various ways ranging from adaptation to environmental challenges and activation of survival pathways to induction of cell death. The initial response to stress encompasses adaptive measures to ensure survival and in the presence of irreparable damage associated with unresolved stress cell death ensues. Understanding the principles and mechanisms governing cell survival over cell death is of particular importance in the field of cancer therapy. It is intriguing that exposure of a seemingly homogenous population to death inducing stimuli, such as chemotherapeutic agents, induces fractional tumor killing in a stochastic manner while a subgroup of cells acquire a persistent state, most probably through activation of compensatory survival pathways. Fractional cell killing and, therefore, inability to completely eradicate transformed cells result in resistance to therapy.
Methods/Results: To gain further insight into compensatory mechanisms and divergent responses elicited in response to death inducing stimuli we designed a multiparametric flow cytometry panel for simultaneous assessment of different forms of cell death at the single cell level, and aimed to dissect stimulus-specific death patterns and pinpoint potential compensatory mechanisms in persistent cells. We modified ( Bergamaschi et al. 2019) and utilized panels including antibodies against RIP3, LC3B, cleaved caspase 3, cleaved PARP-1, PERK, H2AX, p21, Ki-67 and dead cell discriminating dye. This enabled simultaneous interrogation of a multitude of cell death modes including necrosis, necroptosis, apoptosis and parthanatos in response to DNA damage and as well as proliferation, autophagy and endoplasmic reticulum (ER) stress. To test this concept, we initially utilized agents inducing DNA damage and generated two-dimensional t-SNE plots and diffusion maps to illustrate the multifaceted stress response and developmental trajectories upon challenging with DNA damaging agents. Exposure of acute myeloid leukemia (AML) cell lines to etoposide (E) and daunorubicin (DNR) dramatically altered cellular landscape and resulted in emergence of distinct stress responses characterized by differential induction of autophagy, ER stress and DNA damage response and an increase in multiple cell death subpopulations differentially expressing cleaved caspase 3, PARP-1, necrotic cell identifier (live dead aqua dye) and H2AX. We then generated diffusion maps to infer developmental trajectories of dead cells and identified H2AX+PARP+Caspase-3 co-expression as the earliest event occurring in dying cells while cells stained positive for dead cell dye only marked the latest stage. Of note, a fraction of cells exhibited increased autophagy, accompanied with high ER stress and low DNA damage. Presumably, this pattern identifies persistent cells attaining a transient state in response to E and DNR associated with higher likelihood of survival. Evidently, external stress induced a divergent multifaceted response: DNA damage followed by cell death vs. induction of adaptive mechanisms including autophagy and high ER stress.
Although both E and DNR preferentially targeted proliferating cells and induced cell cycle arrest, overall stress response to E was distinct from stress to DNR in high-dimensional plane. To attain a comprehensive overview of stress response to E vs. DNR we compared t-SNE maps depicting overall stress response and observed significant segregation. Autophagy and ER stress was more pronounced in E group while DNR completely abrogated proliferation in surviving cells. To further corroborate the utility of this approach, we assessed the activity of exportin-1 (XPO1, KPT-330) and MDM2 (DS-3032b) inhibitors. KPT-330 and DS-3032b individually induced limited cell death. Combination of XPO-1 and MDM2 inhibitors resulted in enhanced apoptotic cell death with unrepaired DNA damage while surviving cells displayed an autophagy pattern.
Conclusion: These findings provide proof of concept for the utility of single cell mapping of cellular stress in delineating stressor-specific response patterns and identifying potential resistance mechanisms. Single cell mapping of cell stress and cell death can inform the development of more effective combinatorial drug regimens. Studies to identify stress signatures of targeted agents currently developed for the treatment of AML are ongoing
Carter:Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Andreeff:NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; BiolineRx: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Reata: Equity Ownership; Aptose: Equity Ownership.
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